DE60307266T2 - METHODS OF IDENTIFYING AND DEVELOPING COMPONENTS THAT INTERACT WITH POTENTIAL-RELATED KV-4 KINNIC CHANNELS - Google Patents

Abstract

Methods for identifying and developing compounds that interact with voltage-gated potassium channels of the Kv4 family, optionally with auxiliary proteins such as KChiP proteins. Methods for identifying and developing compounds that may interact with voltage-gated potassium channels of the Shal (Kv4) family. Transgenic nematodes of the species Caenorhabditis elegans that are suitable for use in these methods are also described.

Description

The The present invention relates to methods of identification and Development of compounds based on pharmacological and / or veterinary Are useful as well as assays and screens for use with Such methods, and compounds by using this Procedure can be identified.

Especially the invention relates to methods for identifying compounds, those with voltage-dependent potassium channels the Sha1 (Kv4) family (hereafter referred to as "Kv4 channel (channels)") can "interact" (as defined below). These compounds can be added prevention and / or Treatment of diseases or disorders associated with such channels be useful; some of them will be below mentioned.

The The invention also relates to methods for determining whether a compound interact with a Kv4 channel, for example, to determine whether the compound is an agonist or an antagonist (i.e., a blocker or an opener) a Kv4 channel or for a Kv4 channel is.

In some others, non-limiting embodiments The invention relates to assays and screens comprising these methods and / or in the execution these methods can be used; and transgenic roundworms (nematodes), suitable for use with these methods, such as transgenic nematodes the genus Caenorhabditis elegans ("C. elegans").

In a particularly preferred embodiment The invention relates to methods, assays and / or screens suitable for (automated) Screening sentences or libraries of compounds at medium to high throughput are suitable (as more closely below defined) - or configured for it are or can be -. Such methods, assays and / or screens - as well as those identified therewith Connections - for example in the discovery, development and / or production of active Compounds (or compositions containing them) for pharmacological, veterinary and / or agrochemical use, as follows described in more detail becomes.

The The invention therefore also relates to the compounds which are in use the method according to the invention, Assays and / or screens are identified, and use these compounds in the development and / or production of Compositions for pharmaceutical and / or veterinary use.

Further Aspects, embodiments, Applications and advantages of the invention will be apparent from the following Description visible.

Kv4 channels as well their (coding) sequences, their biological function / activity and their Associations with diseases are described in the prior art, See, for example, Bahring et al., J. Biol. Chem., Vol. 276, No. 26, 233888-23894 (2001); Baldwin et al., Neuron 7: 471-483 (1991); Dixon et al. Circ. Res. 79: 659-688 (1996); Dilks et al., J. Neurophysiol. 81: 1974-1977 (1999); Kuo et al., Cell, Vol. 107, 801-813 (2001); anti-tank gun et al., Proc. Natl. Acad. Sci. USA 88; 4386-4390 (1991); Ohya et al., FEBS Lett. 420: 47-53 (1997); Roberts and Tamkun, Proc. Natl. Acad. Sci. USA 88; 1798-1802; Rudy et al., Mol. Cell. Neurosci. 2; 89-102 (1991); Serodio et al., J. Neurophysiol. 75: 2174-2179 (1996); Serodio and Rudy, J. Neurophysiol. 79: 1081-1091 (1998); and Takimoto et al., Circ. Res. 81: 553-539 (1997), as well as therein mentioned further references.

in the Generally, Kv4 channels as voltage-dependent potassium channels involved in membrane depolarization and repolarization processes, e.g. as part of and / or after neuronal discharges and / or as part of the muscle contraction / relaxation cycle.

In particular, as also mentioned in the above references, Kv4 channels are likely to be involved in the native A-type currents generated by different types of primary cells (Dilks et al., Supra), especially in muscle and neuronal cells. Kv4.2 and Kv4.3 transcripts have been found in most neurons, particularly in neurons of the CNS (see Serodio and Rudy, supra, which discuss the distribution of Kv4 channels in rat brains); and heart muscle (see Dixon et al., and Serodio et al., supra, which discuss the occurrence and distribution of Kv4 transcripts in the rat, dog, and human heart). It has also been found that, compared to Kv-type channels from other families, such as Kv1-type channels, the Kv4 channels at potentials below the Activate and inactivate thresholds, with time constants that vary very little as a function of voltage, inactivate (even at very negative potentials), and recover very quickly from inactivation (see Rudy and Serodio, supra).

at neuronal cells, and especially neurons in the brain Kv4 channels among other things probably an important role in the modulation the firing rate, the initiation of activation potentials, shaping burst patterns and postsynaptic signal integration (Dilks et al., And Bahring et al., Supra), and it is believed that they are related to the physiological condition or disorders that from these activities result (Serodio and Rudy, supra).

At the heart, Kv4 channels, among others, play a major role in the calcium-independent A-type currents in the heart muscle (the "transient outward current" or "I _to "), particularly in the ventricular muscle of the heart, and are therefore likely to be at early repolarization and thus the total duration of the action potential and the length of the refractory period (Serodio and Rudy, supra). Therefore, Kv4 channels are believed to be associated with (susceptibility to) cardiac disorders, such as arrhythmias and other types of heart failure (Kuo et al., Supra).

So far were three Kv4 genes in mammals - as Kv4.1 (also known as mSha1), Kv4.2 (also known as RK5) or Kv4.3 marked - cloned and characterized, e.g. in rats and dogs (Dixon et al., Serodio et al., Ohya et al. and Takimoto et al., supra) and at Humans (Dilks et al., And Bahring et al., Supra; see also e.g. WO 98/42833 and US-A-6,395,477).

The Sequences of Kv4 channels in mammals or the genes coding for them are also publicly available Databases like GenBank / NCBI available, e.g. Kv4.1 mouse (accession numbers NP_032449 and A3 8372); Kv4.1 human (accession numbers BAA96454, AAF65617 and RF65516); Kv4.2 mouse (accession number NP_062671 and AAD16972), rat Kv4.2 (Accession number NP_113918); Kv4.2 of humans (accession number AAD22053 and CAB56841); Kv4.3 of the mouse (accession numbers NM_019931 and AF384170), Kv4.3 of the rat (accession number U42975) and Kv4.3 of the human (accession number XM_052127).

The References cited above also suggest that future more channels the Kv4 family can be identified and cloned, for example from neurons of the brain, the Kv4-like O channels, which operate below the threshold, but no high expression of Kv4.1, Kv4.2 and / or Kv4.3 transcripts (see Serodio and Rudy, supra) or other suitable tissues / cells.

As mentioned above, have the Kv4 channels in mammals also a high degree of sequence identity (> 70%) with the Shal-like gene product, which a potassium channel in Drosophila melanogaster (see Baldwin et al., supra, as well as WO 01/58952), and therefore become considered as closely related to these.

The Inventors have also sequenced a gene from C. elegans, the similarity with the Kv4 genes of mammals and which is referred to herein as Kv4.x. The sequence of the gene is given in SEQ ID no. This sequence is different something from those based on the AceDB and EMBL sequence databases (see AceDB # Y73B6BL.19; EMBL # AC084197) predicted sequences.

With Look at the above biological functions and the associations with diseases becomes Expects compounds that act as agonists and antagonists the Kv4 channels (and / or the one with these channels associated biological function (s) and / or metabolic pathways), and, in particular, compounds that can (completely or partially) "block" or "open" Kv4 channels, as pharmaceutically active agents (active ingredients) may be useful, in particular in prevention and / or treatment of heart disease, such as arrhythmias, hypertension-induced Heart disorders, such as hypertension-induced cardiac hypertrophy (e.g., ventricular Hypertrophy), and disorders of the nervous system, such as epilepsy, strokes, traumatic injuries of the brain, states of anxiety, Insomnia, Alzheimer's Disease and Parkinson's syndrome.

Nevertheless, there are no descriptions in the art of methods by which such compounds could (effectively) be identified and / or developed. In particular, no methods are described for screening libraries of chemical compounds for agonists and / or antagonists of Kv4 channels at medium or high throughput (in this regard, it should be noted that the throughput conventional method for screening ion channels, such as the "patch clamp" method or FLIPR method, is quite limited).

It is therefore a general object of the present invention, methods provide with which compounds identified and / or developed can be which can interact with a Kv4 channel, and / or on qualitative and / or quantitative way of determining whether a (particular) compound can interact with or interact with a Kv4 channel.

According to the invention in its broadest interpretation, it was found that this goal was achieved can be made by using - in Such a method, assay or screen - a nematode worm with at least one nucleotide sequence encoding a Kv4 channel, was transformed, e.g. by contacting the roundworm with the / the while to be tested compound (s) in the nematode the Kv4 channel is expressed (i.e., in at least one of its cells, parts, tissues or organs), and then (the change of) at least one detectable / observable (biological) To determine property or peculiarity of nematode, i. when Result of treatment of nematode worm with test (s) Compound (s), i. in comparison with a suitable reference (such as the wild-type or other suitable strain, including, but not limited to the strain used to express the Kv4 channel has been).

In In a first aspect, therefore, the invention relates to a nematode, characterized in that the worm is a heterologous nucleotide sequence expressing a functional voltage-gated potassium channel encoding the Kv4 family, the Kv4.2 or Kv4.3 or a mutant of which with a sequence identity of at least 50% at the amino acid level with the corresponding human Kv4.2 or Kv4.3 channel.

This Roundworm is preferably of the species Caenorhabditis, and is preferably (a strain of) Caenorhabditis elegans. In itself, the threadworm C. elegans wild type (and / or derived therefrom), a suitable Mutant line or strain (and / or derived therefrom), and / or a suitable transgenic line or strain (and / or derived therefrom), as further described below.

Preferably the Kv4 channel (or its coding nucleotide sequence) is heterologous to the nematode used, as further described below is. As such, the Kv4 channel (or its coding nucleotide sequence) of any appro priate biological Source be derived, as further described below. Preferably, the Kv4 channel (or its coding nucleotide sequence) from a mammal derived, in particular preferably from humans.

Also can instead of a course occurring Kv4 channel (or its coding nucleotide sequence) a (natural or synthetic) mutants thereof, as described in more detail below is.

Of the voltage-dependent Potassium channel of the Kv4 family is preferably a Kv4.2 or Kv4.3 channel, wherein a Kv4.3 channel is particularly preferred. The human Kv4.3 coding nucleotide sequence, the used in the experimental part below is in the SEQ ID NO: 4. The amino acid sequence of human Kv4.3 is shown in SEQ ID NO: 5.

Of the Kv4 channel should at least be in at least one cell, one part, be expressed in a tissue or organ of the nematode, as described below. For this purpose, the Kv4 channel coding nucleotide sequence in the worm under the control of a or more suitable regulatory elements are expressed, such as a suitable promoter present in the nematode, as well as below is described.

Preferably leads the Expression of the Kv4 channel to at least one detectable change in or compared to the roundworm used to express this Channel is used. Preferably, the detectable change is which is caused by the expression of the Kv4 channel (below as "detectable phenotypic change ") detectable biological change, and in particular a detectable phenotypic, physiological, Behavioral and / or biochemical change, as described in more detail below becomes.

Particularly preferred is the at least one detectable phenotypic change with the biological activity of the expression of the Kv4 channel in the roundworm (ie in at least one cell, a part, a tissue or organ of the roundworm), eg it is a result of it. Some preferred, non-limiting examples of such detectable phenotypic changes are described below

• (a) in-Kontakt-bringen mindestens eines wie hier beschriebenen Fadenwurms mit der (mindestens einen) Verbindung oder den Verbindungen; und
• (b) Detektieren und/oder Beobachten mindestens einer detektierbaren Änderung des oder in dem Fadenwurm.

In a further aspect, the invention also relates to a method for determining whether a compound or compounds can interact with a voltage-gated Kv4 family potassium channel, the method comprising the steps of:

• (a) contacting at least one nematode as described herein with the (at least one) compound or compounds; and
• (b) detecting and / or observing at least one detectable change of or in the roundworm.

• (a) in-Kontakt-bringen mindestens eines wie hier beschriebenen Fadenwurms mit (mindestens einer) Verbindung oder Verbindungen; und
• (b) Detektieren und/oder Beobachten, ob eine der Verbindungen der Gruppe eine detektierbare Änderung des oder in dem Fadenwurm bewirkt.

The invention also relates to a method for screening a group of compounds, whether one or more of the compounds of this group can interact with a voltage-dependent potassium channel of the Kv4 family, the method comprising the following steps:

• (a) contacting at least one nematode as described herein with (at least one) compound or compounds; and
• (b) detecting and / or observing whether one of the compounds of the group causes a detectable change in or in the roundworm.

• (a) in-Kontakt-bringen mindestens eines wie hier beschriebenen Fadenwurms mit der Verbindung; und
• (b) Detektieren und/oder Beobachten, ob die Verbindung eine detektierbare Änderung des Fadenwurms oder darin bewirkt;

wobei die detektierbare Änderung eine Verbindung identifiziert, die mit dem Kv-Kanal interagieren kann.The invention also relates to a method for identifying a compound capable of interacting with a voltage-gated potassium channel of the Kv4 family, the method comprising the steps of:

• (a) contacting at least one roundworm with the compound as described herein; and
• (b) detecting and / or observing whether the compound causes a detectable change in the nematode or in it;

wherein the detectable change identifies a compound that can interact with the Kv channel.

• (a) in-Kontakt-bringen mindestens eines wie hier beschriebenen Fadenwurms mit der Verbindung; und
• (b) Detektieren und/oder Beobachten, ob die Verbindung eine detektierbare Änderung des Fadenwurms oder darin bewirkt;

wobei die detektierbare Änderung eine Verbindung identifiziert, die bei der Prävention und/oder der Behandlung verwendet werden kann (und/oder die verwendet werden kann, um ein Arzneimittel zur Prävention und/oder Behandlung herzustellen.)The invention also relates to a method of identifying a compound that can be used in the prevention and / or treatment of a disease or disorder associated with or caused by a defect in a Kv4 channel, which method comprises following steps include:

• (a) contacting at least one roundworm with the compound as described herein; and
• (b) detecting and / or observing whether the compound causes a detectable change in the nematode or in it;

wherein the detectable change identifies a compound that may be used in the prevention and / or treatment (and / or that may be used to prepare a medicament for prevention and / or treatment).

The Illness or disorder is preferably a disease or disorder associated with (a disorder of) Kv4.1, Kv4.2 and in particular Kv4.3 or caused by it is, and in particular from the above and / or in the above selected prior art diseases and disorders is selected.

Especially can the disorder or disease associated with a (disorder of) a Kv4 channel (s) or caused heart failure be, such as an arrhythmia, and / or a disease or disorder of the nervous system (including but not limited to on, the central nervous system) associated with (a disturbance) of a Kv4 channel is or is caused by, including, but not limited to to the diseases mentioned above and / or in the above State of the art are described.

Preferably is the detectable change caused by the compound (s) to be tested (hereinafter as "significant biological change ") detectable biological change, and especially a detectable phenotypic, physiological, behavioral and / or biochemical change, as below is described.

In particular, the at least one significant biological change is representative of - ie it is a result of - the interaction of the compound (s) with that expressed in the nematode used Kv4 channel. Some preferred, non-limiting examples are given below.

In In another aspect, the invention relates to the use of a transgenic roundworm, as described in the preceding methods is.

In In another aspect, the invention relates to a compound which using the transgenic nematode and / or those described herein Procedure is identified. These compounds can be agonists and / or antagonists (i.e., blockers or openers) of the relevant Kv4 channel be, and can for example (in the preparation of a pharmaceutical composition) for prevention and / or treatment of diseases or disorders that are used with (faults in) Kv4 channels including, but not limited on the diseases and disorders, the above and / or in the above-mentioned prior art are described.

Further non-limiting Aspects, embodiments, Applications and advantages of the invention will become apparent from the following detailed description of the invention clearly.

Everyone can according to the invention suitable nematode can be used. Preferably, the nematode is a microscopic roundworm, that is, a roundworm with a Size (adult) of not more than 3 mm, preferably not more than 2 mm, and usually between 0.1 and 1.5 mm. Suitable examples are those skilled in the art seen.

• – C. elegans hat einen kurzen Lebenszyklus von etwa 3 bis 4 Tagen.
• Dies bedeutet nicht nur, dass diese Fadenwürmer (und geeignete Mutanten, Transgene und/oder stabile Linien davon) schnell und in großer Anzahl kultiviert/gezüchtet werden können, sondern auch, dass die Verwendung von C. elegans in Testverfahren in relativ kurzer Zeit und mit hohem Durchsatz möglich ist, bei denen die Fadenwürmer in einem oder mehre ren, oder allen, Lebens-/Entwicklungsstadien, und sogar über eine oder mehrere Generationen, getestet werden. Wegen der kurzen Lebensspanne können mit auf C. elegans basierenden Testverfahren Verbindungen in einem oder mehreren, oder allen, Lebens-/Entwicklungsstadien ohne mit der Stabilität und/oder (Bio-)Verfügbarkeit verbundenen Problemen getestet werden. Wegen seiner geringen Größe kann C. elegans auch in den Vertiefungen einer Multi-Well-Platte kultiviert und beobachtet werden, so dass diese Testverfahren automatisiert werden können;
• – C. elegans ist durchscheinend und erlaubt so – vorteilhafterweise – die visuelle oder nicht-visuelle Beobachtung innerer Organe und innerer Prozesse, und auch die Verwendung von Markern, wie fluoreszenten "Reporter"-Proteinen, sogar während der Wurm am Leben ist. Gleichfalls kann, wie nachstehend erläutert ist, diese Beobachtung unter Verwendung geeigneter Ausrüstungen wie Plattenlesegeräten automatisiert vorgenommen werden;
• – Verfahren zur Behandlung, Kultivierung, Erhaltung und Aufbewahrung (z.B. als gefrorene Proben, was große praktische Vorteile bietet) von C. elegans sind im Stand der Technik etabliert, beispielsweise im nachstehend in Bezug genommenen Standard-Handbuch von C. elegans. C. elegans kann beispielsweise als eine oder mehrere Proben mit im wesentlich vollständig isogenem (isogenen) Genotyp(en) verwendet werden;
• – C. elegans ist ein bekannter und gut charakterisierter Modellorganismus. Beispielsweise wurde das Genom von C. elegans vollständig sequenziert (wobei 70% der menschlichen Genlinien konserviert sind), und es sind auch die vollständigen Zelllinien und Zellinteraktionen (beispielsweise von Synapsen) bekannt. Zusätzlich verfügt C. elegans über ein vollständig diploides Genom, und ist in der Lage, sich sexuell zu reproduzieren (z.B. für Kreuzungen) oder sich als selbstbefruchtender Hermaphrodit fortzupflanzen. Weiterhin sind verschiedene Techniken zur genetischen Modifikation von C. elegans im Stand der Technik bekannt, einschließlich von Transformations- und Selektionstechniken. All dies stellt viele Vorteile bereit, nicht nur bei der Verwendung von C. elegans in genetischen und/oder biologischen Untersuchungen, sondern auch bei der Verwendung von C. elegans bei der Entdeckung, Entwicklung und/oder Pharmakologie von (potentiellen) Arzneimitteln zur Verwendung bei Mensch oder Tier.
• – C. elegans kann die zu testenden Verbindungen leicht aus dem umgebenden Medium aufnehmen, beispielsweise über den Gastrointestinaltrakt (z.B. über Pharynxpumpen, gefolgt von aktivem oder passivem Transport durch die Wand des Gastrointestinaltraktes) und/oder durch Diffusion durch die Haut. Auf diesem Weg konnte auch gezeigt werden, dass beim Menschen wirksame Arzneimittel auch eine biologische Wirkung bei C. elegans haben (d.h. auf den biologisch relevanten Phänotyp).

oder jede Kombination davon.In particular, the roundworm used belongs to the genus Caenorhabditis, and in particular it is Caenorhabditis elegans. In this embodiment, the invention provides one or more advantages associated with the use of C. elegans, such as:

• C. elegans has a short life cycle of about 3 to 4 days.
• Not only does this mean that these roundworms (and suitable mutants, transgenes and / or stable lines thereof) can be cultured / grown rapidly and in large numbers, but also that the use of C. elegans in test procedures in a relatively short time and with high throughput is possible in which the roundworms are tested in one or more ren, or all, stages of life / development, and even over one or more generations. Because of the short lifespan, C. elegans-based assays can test compounds at one or more, or all, stages of life / development without problems associated with stability and / or (bio) availability. Because of its small size, C. elegans can also be cultured and observed in the wells of a multi-well plate so that these assays can be automated;
• - C. elegans is translucent, thus allowing - advantageously - the visual or non-visual observation of internal organs and processes, as well as the use of markers such as fluorescent "reporter" proteins, even while the worm is alive. Likewise, as explained below, this observation can be automated using suitable equipment such as plate readers;
• Methods of treatment, cultivation, maintenance and storage (eg as frozen samples, which offers great practical advantages) of C. elegans are well established in the art, for example in the standard manual of C. elegans, referred to below. For example, C. elegans may be used as one or more samples having substantially completely isogenic (isogenic) genotype (s);
• C. elegans is a well-known and well-characterized model organism. For example, the genome of C. elegans has been completely sequenced (with 70% of the human gene lines conserved), and complete cell lines and cell interactions (for example, synapses) are also known. In addition, C. elegans has a fully diploid genome, and is able to reproduce sexually (eg, for crossbreeding) or reproduce as a self-fertilized hermaphrodite. Furthermore, various techniques for the genetic modification of C. elegans are known in the art, including transformation and selection techniques. All this provides many benefits, not only in the use of C. elegans in genetic and / or biological studies, but also in the use of C. elegans in the discovery, development and / or pharmacology of (potential) drugs for use Human or animal.
• C. elegans can easily ingest the compounds to be tested from the surrounding medium, for example via the gastrointestinal tract (eg via pharyngeal pumps, followed by active or passive transport through the wall of the gastrointestinal tract) and / or by diffusion through the skin. In this way it could also be shown that drugs effective in humans also have a biological effect on C. elegans (ie on the biologically relevant phenotype).

or any combination thereof.

To the easier understanding In the following, the invention will be described with reference to C. elegans. From the foregoing, however, it will be apparent that the invention in their broadest interpretation, not to the use of this specific Nematode worm is limited.

For general Information about C. elegans and methods of treating this roundworm is going to the standard manuals too C. elegans, for example W.B. Wood et al., "The nematode Caenorhabditis elegans, "Cold Spring Harbor Laboratory Press (1988); D.L. Riddle et al., "C. ELEGANS II", Cold Spring Harbor Laboratory Press (1997); "Caenorhabditis elegans, Modern Biological analysis of an organism ", edited by H. Epstein and D. Shakes, Methods in Cell Biology, Vol. 48, 1995; and "C. elegans, a practical approach", ed. from I.A. Hope, Oxford University Press Inc, New York, USA, 1999.

In addition will be some other methods and methods for performing in vivo assays using C. elegans in the applicant's applications below described: PCT / EP99 / 09710 (published on June 15, 2000 as WO 00/34438); PCT / EP99 / 04718 (published on January 15, 2000 as WO 00/01846); PCT / IB00 / 00575 (published on October 26, 2000 as WO 00/63427); PCT / IB00 / 00557 (published on 26.

October 2000 as WO 00/53425); PCT / IB00 / 00558 (published October 26, 2000 as WO 00/63426); and, for example, in PCT / US98 / 10080 (published on November 19, 1998 as WO 98/51351), PCT / US99 / 13650, PCT / US99 / 01361 (released on July 29, 1999 as WO 99/37770), and PCT / EP00 / 05102.

Everyone can according to the invention suitable strain or line of C. elegans with one or more transformed several Kv4 channel coding nucleotide sequences and / or may otherwise be used to express a Kv4 channel be arranged so that a roundworm is provided, the for the use according to the invention suitable. Suitable lines or strains are familiar to the expert, and can (without restriction) Wild-type and N2 strains as well as mutants and / or transformed lines or strains of C. elegans, including, but not limited to those in the standard manuals above C. elegans listed mutant lines or strains.

• – Wildtyp- und allgemein verwendete Laborstämme wie N2 und Hawaii;
• – Stämme mit konstitutivem Pumpen, insbesondere unc-31 und HD8 (LMBP 5447CB, auch bg46 oder hdr (bg46) genannt, siehe die PCT/IB00/00557 des Anmelders). Unter diesen Stämmen/Mutanten kann jedes geeignete Allel verwendet werden, beispielsweise unc31 (e928).
• – unc Mutanten, insbesondere unc-19, unc-36 und unc-119. Unter diesen Mutanten kann jedes geeignete Allel verwendet werden, beispielsweise unc36(e251) oder unc-119(ed3).
• – egl-Mutanten, insbesondere egl-16, egl-19, egl-36 und egl-139. Unter diesen Mutanten kann jedes geeignete Allel verwendet werden, beispielsweise egl-19(n582), egl-36(sa577), egl-36(n728) oder egl19(n2368).
• – eat-Mutanten, insbesondere eat-16. Unter diesen Mutanten kann jedes geeignete Allel verwendet werden, beispielsweise eat16(sy438).
• – exp-Mutanten, insbesondere exp-2-Mutanten, und insbesondere bevorzugt exp-2-Mutanten mit Funktionsverlust. Unter diesen Mutanten kann jedes geeignete Allel verwendet werden, beispielsweise exp-2(sa26ad1426) oder exp-2(sa26ad1201)(für letzteres siehe Davis, Dent und Avery, Worm Breeder's Gazette 14(4):72 (1996).
• – pha-Mutanten, insbesondere pha-1. Unter diesen Mutanten kann jedes geeignete Allel verwendet werden, beispielsweise pha-1 (e2123).

Some preferred C. elegans lines or strains for use in the present invention include, but are not limited to:

• - wild-type and commonly used laboratory strains such as N2 and Hawaii;
• Constitutive pumping strains, in particular unc-31 and HD8 (LMBP 5447CB, also called bg46 or hdr (bg46), see Applicant's PCT / IB00 / 00557). Among these strains / mutants, any suitable allele may be used, for example, unc31 (e928).
• - unc mutants, especially unc-19, unc-36 and unc-119. Among these mutants, any suitable allele may be used, for example, unc36 (e251) or unc-119 (ed3).
• Egl mutants, in particular egl-16, egl-19, egl-36 and egl-139. Among these mutants, any suitable allele may be used, for example, egl-19 (n582), egl-36 (sa577), egl-36 (n728), or egl19 (n2368).
• - eat mutants, especially eat-16. Among these mutants, any suitable allele may be used, for example, eat16 (sy438).
• Exp-mutants, especially exp-2 mutants, and particularly preferably exp-2 mutants with loss of function. Among these mutants, any suitable allele may be used, for example, exp-2 (sa26ad1426) or exp-2 (sa26ad1201) (for the latter see Davis, Dent and Avery, Worm Breeder's Gazette 14 (4): 72 (1996).
• Pha mutants, especially pha-1. Among these mutants, any suitable allele may be used, for example pha-1 (e2123).

So are two preferred but non-limiting classes of mutants the "muscle-related" mutants (such as exp-2, unc-19 and other muscle-related mutants mentioned above), and the "neuron-related" mutants.

A preferred, but non-limiting group of mutants for expression of the Kv4 channel according to the invention include those in which one or more of the native ion channels involved in neuronal functions, and in particular one or more of which in polarization and / or repelization of neuronal and / or muscle membranes (including, but not limited to, Kv4 channel orthologs in the nematode, such as Kv4.x and other native ion ka Nalen ion channels have been changed compared to the wild type or N2 (for example, downregulated or completely switched off (knocked out)). For example, these may be mutants in which one or more of the potassium channels involved in membrane repolarization are fully or partially closed (either transiently, as in RNAi, or constitutive, such as by an appropriate mutation). In the latter mutants - of which the exp-2 mutant is a preferred but not limiting example - expression of the Kv4 channel may partially or completely restore or "rescue" the mutant phenotype. In general, such mutants can be identified by means of an electropharyngogram ("EPG") and / or intracellular electrical recordings ("IER") of the pharyngeal muscle, since their EPG and / or IER distinguishes them from the wild type or N2 EPG and / or IER ,

Regarding "electropharyngograms" ("EPG") and "intracellular electrical Notes "(" IER ") see Raizen and Avery, Neuron, Vol. 12, 483-495 (1994) and the aforementioned C. elegans standard manuals, especially the chapter of Avery and Thomas in Riddle et al., C. ELEGANS II (Chapter 24, pages 679-716) and Hope et al., In particular Pages 171-176. Alternatively, the neuronal or muscle activity may be below Use of well-known patch-clamp method can be determined.

For example, mutants such as exp-2 in the EPG have a "repolarization" (or "relaxation") peak (peak "R", see Avery and Thomas, 5 on page 688), which is reduced or even substantially absent from N2, and / or they have a "refractory" (or "relaxation") period (ie, the time between the excitation peak "E" and the Relaxation peak "R", sometimes also referred to herein as "plateau phase", "P-phase" or "duration", both below and in the art), which is altered from N2 in the wild type See also Davis, Dent and Avery, Worm Breeder's Gazette 14 (4): 72 (October 1, 1996), this reference shows comparative wild-type EPG's and exp-2 mutants, as well 1 showing the EPG of an N2 background expressing a human Kv4.3 channel under the control of a myo-2 promoter (according to the invention).

In a preferred, but non-limiting embodiment The invention features the strains used to express the Kv4 channel reduced pharyngeal pumping and / or reduced drinking behavior (see below), preferably both, compared to wild-type / N2. Because of defects in membrane repolarization, the exp-2 mutant is also a non-limiting Example of this.

In a further embodiment For example, the strains used to express the Kv4 channels show one decreased sensitivity and / or activity of (one or more) sensory neurons. A preferred, but non-limiting Example of such a "sensory Neuron-related "mutant strain is osm-3.

The above and other mutants (and their suitable alleles) which used according to the invention can, are generally described in the prior art - for example in the above C. elegans standard manuals - and can be found, for example, by Caenorhabditis Genetics Center ("CGC"), St. Paul, Minnesota, USA, related.

Of the Kv4 channel (or its coding nucleotide sequence) used for transformation of nematode worm, any voltage-gated potassium channel can be used (or the nucleotide sequence encoding it) of the Kv4 family. It may therefore be (a coding nucleotide sequence for) one Kv4.1, Kv4.2 or Kv4.3 channel, or any other voltage-dependent Kv4 potassium channel, which is described in the prior art or the after the filing date of the present application.

Preferably the Kv4 channel is Kv4.2 or Kv4.3, where Kv4.3 is very particularly preferred.

Of the Kv4 channel (the nucleotide sequence encoding it) may be any suitable one obtained from a biological source; usually this is a multicellular, in particular, a multicellular cell containing one or more neuronal cells and / or muscle cells.

• – anderen Wirbeltieren, einschließlich, aber nicht beschränkt auf, Vögel, Fische, wie beispielsweise Zebrafische, Reptilien und Amphibien wie beispielsweise Xenopus;
• – Wirbellosen, einschließlich, aber nicht beschränkt auf Insekten, wie beispielsweise Drosophila, Heliothus, Hausfliege, Moskito, oder Aphidae (z.B. Sha-l-ähnliche Kanäle), Fa denwürmer, wie C. elegans (z.B. Kv4.x-Kanäle), und schädliche und/oder parasitäre Fadenwürmer, wie beispielsweise Ostertagia, Haemonchus und Trichostringylus.

Preferably, the Kv4 channel (the nucleotide sequence encoding the Kv4 channel) is from a vertebrate, in particular from a warm-blooded animal, and most preferably from a mammal, such as a rat, a mouse, a rabbit, a sheep, a pig , a goat, a dog, a non-human primate or a human (this is particularly preferred). However, the invention in its broadest sense is not limited thereto, but includes, for example, the use of Kv9 channels:

• - other vertebrates, including, but not limited to, birds, fish such as zebrafish, reptiles and amphibians such as Xenopus;
• Invertebrates, including, but not limited to, insects such as Drosophila, Heliothus, house fly, mosquito, or Aphidae (eg, Sha I-like channels), fa worms, such as C. elegans (eg, Kv4.x channels), and harmful and / or parasitic nematodes such as Ostertagia, Haemonchus and Trichostringylus.

• – (einer oder mehreren Nukleotidsequenaen, kodierend) ein oder mehr Teile oder Fragmente eines natürlich vorkommenden Kv4-Kanals; und/oder mit
• – (einer oder mehreren Nukleotidsequenzen, kodierend) eine natürliche oder synthetische Mutante, Variante, ein Analog, Homolog oder Ortholog eines natürlich vorkommenden Kv4-Kanals, und/oder eines oder mehrere Teile oder Fragmente davon;

die hier alle gemeinsam als "Kv4-Kanal -Mutant(en)" bezeichnet werden. Diese Kv4-Kanal-Mutanten können – beispielsweise – sich von einem natürlich vorkommenden Kv4-Kanal durch Addition, Substitution, Insertion und/oder Deletion einer oder mehrerer Aminosäurereste (oder, im Fall einer Nukleotidsequenz, einer oder mehrerer Basen/Nukleotide) in einer oder mehreren Positionen unterscheiden.In the context of the invention, the roundworm can also be transformed with:

• - (one or more nucleotide sequences encoding) one or more parts or fragments of a naturally occurring Kv4 channel; and / or with
• - (one or more nucleotide sequences encoding) a natural or synthetic mutant, variant, analogue, homologue or orthologue of a naturally occurring Kv4 channel, and / or one or more parts or fragments thereof;

which are all collectively referred to as "Kv4 channel (s)". These Kv4 channel mutants may, for example, be derived from a naturally occurring Kv4 channel by addition, substitution, insertion and / or deletion of one or more amino acid residues (or, in the case of a nucleotide sequence, one or more bases / nucleotides) in one or more different positions.

Preferably is any such Kv4 channel mutant from a naturally occurring mammalian Kv4 channel derived, and in particular from a naturally occurring human Kv4 channel. For example, you can natural Ortholay of human Kv4 channels of other species using bioinformative methods and / or by the use of probes / primers based on the human Kv4 sequence be identified.

Preferably also includes any used in the invention Kv4 channel mutant (the nucleotide sequences encoding it) sequence identity of at least 50%, preferably at least 60%, especially preferred at least 70%, most preferably at least 80%, most preferably at least 90%, and more preferably at least 95% the level of amino acids with the corresponding human Kv4 channel (e.g., Kv4.1, Kv4.2 and preferably Kv4.3) (or, alternatively, with the Kv4.x sequence of SEQ ID NO: 3). Therefore may be the percentage of "sequence identity" between the amino acid sequence a Kv4 channel mutant and the amino acid sequence of a human Kv4 channel calculated by [the number of amino acid residues in the sequence of Kv4 channel mutant those with the amino acid residues at the corresponding amino acid position the amino acid sequence of the human Kv4 channel are identical] by [the total number of amino acid residues in the amino acid sequence of the human Kv4 channel] and multiplied by [100%], wherein each deletion, insertion, substitution or addition of a amino acid residue in the sequence of the mutant - i. compared to the given sequence - as a difference in the amino acid position is looked at. Alternatively, the degree of sequence identity may be below Using a known computer algorithm for sequence matching, such as NCBI Blast v2.0, calculated using standard constraints become.

To this purpose is the amino acid sequence of human Kv4.3 in SEQ ID No. 5. Regarding the Sequences of other human Kv4 channels to the above-mentioned state directed to the technique.

Used according to the invention Naturally occurring Kv4 channel mutants can from a suitable biological source in a conventional manner be identified and isolated, for example by direct Expression from a DNA library, by screening a DNA library with a suitable probe, or by isolation of an appropriate mRNA, followed by cDNA synthesis using reverse transcriptase.

For example, synthetic Kv4 channel mutants can be obtained, eg, based on and / or from the sequence of a naturally occurring Kv4 channel, by methods such as automated DNA synthesis, site-directed mutagenesis, combination of two or more portions of one or more natural occurring Kv4 channels (eg to provide a "hybrid Kv4 channel"), introduction of mutations resulting in expression of a truncated expression product, or introduction of mutations by a polymerase chain reaction (PCR reaction) using a or multiple "mismatched" primers. These and other methods are known to those skilled in the art; For example, reference is made to the standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989), and F. " Au subel et al., Eds., "Current Protocols in Molecular Biology," Green Publishing and Wiley Interscience, New York, USA (1987).

As already indicated above, it is further provided that more to the Kv4 family belonging channels identified and / or the date of filing the present application (further) be characterized (for example in mammals and especially in humans), for example, as a result of functional Genome programs and / or targeted search programs. It will expects the invention to be equally - i. analogous to the one described here Way - up these new Kv4 channels can be applied, i. for identification and development of connections that can interact with these channels and / or to determine if a connection is interacting with these channels can.

Preferably the Kv4 channel (the nucleotide sequence encoding it) is "heterologous" to the nematode used, by which is meant that the Kv4 channel (the nucleotide sequence coding it) in the threadworm naturally / natively does not occur (and where the term "hetero-log" is intended to mean: (a) synthetic Kv4 channels, such as described here; and (b) mutants, variants, analogs, homologs, Orthologues, parts and / or fragments of the Kv4 channels that were used natively in the study Threadworm occur). In its broadest sense is the invention but not limited to this and also includes the use of homologous Kv4 channels (i.e. naturally occurring in the nematode used); that can be one such native Kv4 channel - e.g. "Kv4.x", to which reference is taken; see SEQ ID NO: 3 - for example overexpressed (i.e., compared to the native strain used), for example under the regulation of a promoter with which this channel is native is not associated.

Even though it is preferred according to the invention is that the roundworm is transformed with a single Kv4 channel is, the threadworm in the context of the invention, where appropriate also transformed with two or more different Kv4 channels and / or optionally with one or more Kv4 channels and one or more other heterologous genes, including, but not limited to one or more components of the (metabolic) pathway (s), where the Kv4 channel is (probably) involved, and / or one or more Hilfsproteinen, acceptor molecules and / or Subunits of Kv4 channels, including, but not limited on KChiP's, like for example KChiP1, KChiP2 and KChiP3, as well as Kvβ (see for example Bahring et al., Below; and Kuo et al., supra, and An et al., Nature, Vol. 403, pp. 553-556, (2000)). The one or more heterologous gene (s) (or the nucleotide sequence (s) encoding them) can again, of course occur (e.g., from the same biological source as the Kv4 channel and / or out another suitable biological source), and / or they may be synthetic Sequences, finally Mutants, parts, fragments, etc. as described above.

in the Generally can which encode the Kv4 channel (the Kv4 channels) Nucleotide sequence (s) may be genomic sequences (the native introns can contain), and / or cDNA sequences, with cDNA sequences being preferred. These Nucleotide sequences can also - for example - artificial Contain introns, and they can Donor / acceptor splice sites included to make changes to maximize heterologous transgenic expression, and / or they can include an adapted codon for expression in the roundworm to increase.

the Professional is also clear that during / after the expression of the Kv4 channel coding sequence (s) in the Roundworm - and dependent on from the specific Kv4 coding sequence (s) and the particular used nematode - a Gene splicing, SL1 / SL2 splicing, and / or post-translational modifications may occur as well also an intracellular Transport (especially for localization on or at the cell surface / cell membrane). All these steps are encompassed by the invention, provided that that a "functional Kv4 channel "received becomes; By this is meant generally a Kv4 channel, which - according to the as described here - on interaction with those of interest Compound (s) can be tested, i. as part of the process (s) according to the invention or assays.

Of the Kv4 channel (or one or more genes coding for it) can be used for use according to the invention as a single nucleic acid (sequence) which is generally preferred, or as two or several separate nucleic acids.

In general, the Kv4 channel is expressed in the roundworm by having the roundworm (or at least one cell, tissue, organ or part thereof, including, but not limited to, gonads and / or OoCytes) one or more of the desired Kv4 -Channel encoding nucleic acid (s), after which the transformed roundworm (or a descendant) is exposed to conditions / un conditions in which the expression of the Kv4 channel is obtained, ie in at least one part, tissue, organ or a cell of the roundworm.

Preferably are the Kv4 channel encoding nucleic acids in the form of a genetic Construct, which may be DNA or RNA, and preferably as double-stranded DNA. The construct may also be in a form that is for transformation the nematode worm is suitable, in a form suitable for Integration into the genomic DNA of the nematode used is, in a form intended for independent replication, conservation and / or heredity in the nematode worm used (e.g. as part of the extrachromosomal assembly), and / or in a mold, to the independent Replication, preservation and / or inheritance in another desired Host organism (such as a microorganism used for cloning, e.g. E. coli) is suitable.

For example For example, the genetic construct may be in the form of a plasmid, vector, transposon, and / or linear fragments, such as linear PCR fragments.

The (the) used in the invention genetic construct (s) can furthermore - i. in addition to the Kv4 channel encoding one or more nucleic acid sequence (s) - an or several other, known per se, suitable elements of a genetic Construct, including, but not limited suitable regulatory elements (such as a suitable promoter, Enhancer, terminator, etc .; these regulatory elements can too used to regulate the expression of the Kv4 channel in the nematode be), 3'- or 5 'UTR sequences, leader sequences, Selection markers, expression marker / reporter genes, splice / donor sites, and / or elements representing the (rate of) transformation or integration facilitate or increase.

These and other suitable elements for Genetic constructs are familiar to the person skilled in the art, and can be obtained, for example, from the Depend on the type of construct used and the nematode to be transformed; of the cell (s), tissue (s), part (s) or organ (s), in which (which) the Kv4 channel is to be expressed; the way in which the Kv4 channel expresses should be (for example, over constitutive, transient or inducible expression); and or the transformation method used. Some specific, but non-limiting Examples of such elements for use with roundworms, and in particular in C. elegans, are listed below. Other suitable elements can for example, in the above-mentioned standard manuals looked up (e.g., Sambrook et al., and Ausubel et al.), supra cited C. elegans standard manuals (e.g., Wood et al., Riddle et al., Epstein and Shakes, and Hope), and in the above Applicant's applications, as well as in other patent literature (see for example, WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, and / or WO 98/21355).

Preferably are in the gene constructs of the invention the one or more optional element (s) with the one (s) the Kv4 channel-encoding nucleotide sequence (s) and / or with each other "operably linked"; that is general meaning that they are functionally related to each other. For example, one considers a promoter to be "operably linked" to a coding one Sequence if the promoter is transcription and / or expression initiate or otherwise control / regulate a coding sequence (it being understood that the coding sequence is "under the control" of the promoter). When two nucleotide sequences are operably linked, they are generally in the same orientation and usually also in the same reading frame. Usually they also merge, though this is not necessary have to be.

Preferably are the optional further elements of the invention (s) used Genkonstrukte so that they their intended provide biological function in the nematode. For example should be a promoter, an enhancer (enhancer), and / or a terminator to be "operable" in the roundworm, by which means is that (for example) the promoter - at least in one cell, one Tissue, an organ or part of the nematode - the transcription and / or the expression of a nucleotide sequence - e.g. a coding sequence - with whom he operably linked is (as defined above), initiate or otherwise can control / regulate.

• – induzierbare, spezifische Promotoren wie beispielsweise: CYP35A2, CYP35A3 mtl-1, mtl-2
• – konstitutive, spezifische Promotoren wie beispielsweise: ceh-28, dpy-7, egl-15, ges-1, glh-2, glp-1, mec-7, myo-2, myo-2 min., myo-3, osm-6, pes-10 min., pIRES, serca, unc-119, unc-129 full, unc-129-BstEll frag, unc-18, unc-183, unc-4, unc-27, unc-49, unc-54 Enhancer/Promotor, unc-71, unc-8, xol-1
• – induzierbare, nicht-spezifische (d.h. "ubiquitäre") Promotoren, wie beispielsweise: hsp-16.2, hsp-16.41, hsp-16.48, das Tet-ON/Tet-Off^®-Promotorsystem
• – konstitutive, nicht-spezifische Promotoren, wie beispielsweise: let-858
• – vorübergehende Promotoren, wie beispielsweise vit-2.

Such a promoter may be a constitutive promoter or an inducible promoter and may be homologous (which is usually preferred) or heterologous to the nematode used. Some non-limiting examples of suitable promoters include, but are not limited to:

• Inducible specific promoters such as: CYP35A2, CYP35A3 mtl-1, mtl-2
• Constitutive, specific promoters such as: ceh-28, dpy-7, egl-15, ges-1, glh-2, glp-1, mec-7, myo-2, myo-2min, myo-3, osm-6, pes-10min, pIRES, serca, unc-119, unc-129 full, unc-129-BstEll frag, unc-18, unc 183, unc-4, unc-27, unc-49, unc-54 enhancer / promoter, unc-71, unc-8, xol-1
• - inducible, non-specific (ie "ubiquitous") promoters, such as: hsp-16.2, hsp 16:41, 16:48 hsp, the Tet-On / Tet-Off ^® promoter system
• - constitutive, non-specific promoters, such as: let-858
• Temporary promoters such as vit-2.

Further suitable heterologous promoters used for C. elegans can, can be found in the C. elegans standard manuals and in the above Registrations of the applicant.

• – Muskelgewebe (beispielsweise der sca-1 Promotor), insbesondere auf den Pharynxmuskel (beispielsweise der myo-2 Promotor); Körperwandmuskel (beispielsweise der myo-3 Promotor); und/oder der Vulvamuskel (beispielsweise der egl-15 oder egl-35 Promotor);
• – Neurone (beispielsweise der unc-18 oder unc-119 Promotor), und insbesondere: (a) die M4-Neurone, welche inter alia den Isthmus kontrollieren (beispielsweise der ceh-28 Promotor), und/oder (b) die sensorischen Neurone (beispielsweise ASI-spezifische Promotoren wie die daf-7, gpa-4 oder str-3 Promotoren; AWA-spezifische Promotoren, wie die odr-7, odr-3, odr-4, odr-10, osm-9, gpa-5 oder gpa-6 Promotorens AWB-spezifische Promotoren, wie die str-1, gcy-10, odr-1 oder tax-2; oder andere geeignete odr, gpa, osm, str oder tax Promotoren);
• – den Darm (beispielsweise der vit-2 Promotor).

Preferred promoters according to the invention are promoters which direct (at least) the expression of the Kv4 channel

• Muscle tissue (for example the sca-1 promoter), in particular the pharyngeal muscle (for example the myo-2 promoter); Body wall muscle (e.g., the myo-3 promoter); and / or the vulvar muscle (e.g., the egl-15 or egl-35 promoter);
• Neurons (for example the unc-18 or unc-119 promoter), and in particular: (a) the M4 neurons which inter alia control the isthmus (for example the ceh-28 promoter), and / or (b) the sensory neurons (For example, ASI-specific promoters such as the daf-7, gpa-4 or str-3 promoters; AWA-specific promoters such as the odr-7, odr-3, odr-4, odr-10, osm-9, gpa- 5 or gpa-6 promoters AWB specific promoters, such as the str-1, gcy-10, odr-1 or tax-2, or other suitable odr, gpa, osm, str or tax promoters);
• The intestine (for example the vit-2 promoter).

One another promoter which can be used according to the invention, in particular for expression in neuronal cells and / or muscle cells, is the Promoter of the native C. elegans Kv4.x sequence, to which reference is hereby made taken (or an operable fragment thereof),

A another class of suitable promoters are the troponin promoters, including, but not limited on the troponin C, the troponin I and / or the troponin T promoter, including, but not limited on Tnl-1, Tnl-2, Tnl-4, TnC or pat-10, TnT-1 or TnT-2, the for consistent expression in muscle tissue (such as the pharyngeal muscle, the body wall muscle, vulvar muscle, anal muscle), either while the entire life cycle or during specific stages thereof (e.g., embryo and larvae for Tnl-1, and larvae and adults for TnI-2).

One Selection marker should be the threadworms that make up the genetic construct contain, distinguish - e.g. enable their detection and / or selection. A selection marker For example, any gene that can be used to be - under suitable conditions, such as the use of a appropriate selection medium, or a suitable temperature - to select the roundworms, the - e.g. as a result of a (successful) transformation, inheritance or Crossing - that have the genetic construct containing the marker.

Some preferred but non-limiting examples for Selection markers suitable in / for the selection of C. elegans, include pha-1. Other suitable selection markers for use in C. elegans are known in the art; it will, for example Reference is made to the C. elegans standard manuals and the above Registrations of the applicant.

A leader sequence (secretory leader) should be such that - concern in the nematode or in the _n the cell (s), tissue (s), organ (s) or part (s) - the desired post-translational modifications allowed; it may direct the mRNA and / or the translated amino acid sequence to a desired part or organelle of the cell; and / or it can control the secretion of the expression product from the cell. For example, it can represent any pro-, pre, or pre / pro sequence that is operable in the cell or organism.

Some preferred but non-limiting examples of leader sequences for use in C. elegans include, for example, the daf-2 leader sequence, the leader sequence of the Kv4.x gene described herein, and / or the C. elegans splice leader sequences ("SL sequences"). ). Other suitable leader sequences for use in C. elegans are known to those skilled in the art; For example, reference is made to the C. elegans standard manuals and Applicant's aforementioned applications. In this regard, it should also be said that for the expression of a Kv4 channel, a leader sequence that has the necessary posttranslatio and / or which directs the expressed Kv4 channel to the cell membrane, although not necessary, but often more preferred.

One Expression marker or reporter gene should be such that it - in the roundworm or its cell (s), tissue (s), organ (s) or part (s) - the Expression of the genetic construct to be detected (a gene or a nucleotide sequence present on it), and he should optionally also the localization of the expression product allow, e.g. in a specific cell (s), tissue (s), organ (s) or part (s) of nematode, or in specific parts or organelles such cells (like - im Case of present Kv4 channels - in or on the cell membrane). Such a reporter gene can also be called a merger of proteins with one (at least part of) a Kv4 channel (s) are expressed.

Some preferred but non-limiting Examples of reporter genes / expression markers for use in C. elegans include, for example, in the prior art Technique well-known fluorescent proteins, such as the naturally occurring and / or commercially available "GFPs". Other suitable Reporter genes for use in C. elegans are known in the art; For example, reference is made to the C. elegans standard manuals and the aforementioned applications of the applicant.

suitable Examples of other elements of genetic constructs with the constructs of the invention can be used - such as Terminators, transcriptional and / or Translational Enhancer (Amplifier) and / or integration factors - are also known to those skilled in the art and include, but are not limited to, Terminates, such as a polyA sequence or a 3 'UTR (e.g. derived from C. elegans such as the unc-54 3 'UTR), enhancers such as UL6, and Integration factors, such as the C. elegans SL sites and C. elegans outrons; again reference is made to the C. elegans standard manuals and the above-mentioned applications of the applicant.

According to the invention, the the Kv4 channel encoding nucleotide sequence with at least one other Sequence operably linked be that another amino acid sequence, such as a protein or polypeptide encoded so that - according to the Expression - one Protein fusion of the Kv4 channel with the further amino acid sequence is obtained. For example, the further sequence may be a specific one and / or desired biological activity or can provide a reporter sequence, such as a GFP.

The constructs according to the invention can be provided in a conventional manner; in general are procedures such as restriction and linkage of Nucleic acids / nucleic acid sequences involved, as is known in the art. It becomes reference again Taken on the standard manuals, how For example, Sambrook et al. and Ausubel et al., supra mentioned were. The in the constructs of the invention existing additional elements can from a suitable biological source, one of the prior art described vector or plasmid, or may be isolated Use of well-known nucleic acid synthesis methods synthesized become.

Of the Nematode can be infected with (the genetic construct containing the) heterologous Kv4 channel-encoding nucleic acid (s) in any suitable Be transformed by the threadworm used and from some further considerations mentioned above regarding the used genetic constructs may depend. Some preferred, but non-limiting Methods of transforming C. elegans include microinjection, Irradiation (e.g., with gamma radiation), ballistic transformation, Soaking, Feeding, Electroporation, or any other suitable and known per se Transformation method, with microinjection and ballistic Transformation (e.g., to create integrated lines) especially are preferred. These methods, in particular microinjection, may also be microinjection into the oocyte or gonad of a (parent) worm to include the nucleic acid (s) to introduce into the offspring. For further description, turn to the aforementioned C. elegans standard manuals reference taken.

The Transformation of C. elegans may also be more specific mutant strains or lines which are particularly suitable / adapted to the Transformation, integration and / or the specific used Transformation / integration process are. A preferred but non-limiting example of this is unc-119, which especially for the ballistic transformation is appropriate, and of which each suitable allele can be used, such as unc-119 (ed3).

To the transformation can (can) the Kv4 channel coding nucleic acid (s) into the genome of the roundworm (preferably in a stable manner, e.g. to provide an integrated line that sometimes works in practice may be preferred) or they can be obtained and / or substantially independent be inherited (again preferably in a stable manner), for example as part of the extrachromosomal arrangement.

A another method of making a nematode worm, the Kv4 channel may be the crossing of a first roundworm, the the Kv4 channel (the nucleic acid (s) encoding it) (e.g. integrated into the genome), with another roundworm of the same Species to produce offspring that at least the Kv4 channel coding nucleic acid (s) and most preferably also the Kv4 channel below express suitable conditions as described below is. Suitable methods for crossing and subsequent selection from C. elegans - ie to provide offspring that have one or more interested Have properties such as the expression of a Kv4 channel - are the Specialist known. In general, the above-mentioned C. elegans standard manuals Referenced.

Corresponding can in the process according to the invention not just one obtained directly as a result of a transformation Roundworm are used, but also more generations, descendants and Offspring thereof (these are included in the term "threadworm" as used herein). In a preferred, but non-limiting embodiment These are additional generations, descendants and descendants Part of and / or represent a stable line or a stable one Strain, i. a strain that expresses the Kv4 channel (express can).

to Expression of the heterologous Kv4 channel may be the nematode in general cultivated, bred and / or under conditions where the Kv4 channel is expressed becomes. In general, these terms are from the one used Threadworm dependent and of the regulatory elements affecting the expression of the Kv4 channel control, and can the presence of a suitable food source or nutrients include (although it may sometimes be beneficial to the organism while a certain period of time, one or more of these nutrients or deny feed components), and / or other factors which are necessary for the growth or maintenance of roundworms; a suitable medium, a suitable temperature, and the presence a suitable inducing factor or compound (i.e. if the Kv4 channel coding nucleotide sequence of the control of an inducible promoter subject). In general, these conditions are those skilled in the art common; it will turn to the aforementioned C. elegans standard manuals and the aforementioned Applications of the applicant referred.

For example can suitable conditions include the use of a suitable liquid, solid, semi-solid or viscous medium, for example Agar plates, M9, S buffer or a viscous medium with a viscosity higher than M9 (e.g., as in PCT / IB00 / 00575 and other applications of the applicant described, and e.g. using an Ostwald, Ubbelohde or Brookfield viscometer at the culture and / or incubation temperature measured) on a plate, in a Petri dish, in a tube, in a flask, in a well of a multi-well plate, or in another suitable vessel or container; at a temperature between 15 and 30 ° C (usually about 20 to 25 ° C), and in the presence of a suitable food source, such as Bacteria - e.g. E. coli - in in an amount between 0.05 and 0.5 wt .-%, preferably about 0.125 wt .-%.

Under In these conditions, the Kv4 channel can be constitutively expressed (which usually preferred), temporarily (for example, only during a specific phase in the life cycle of roundworms), and / or only if, depending on from the promoter used, a suitable induction takes place. In any case, the Kv4 channel should be in at least one cell, one Tissue, an organ or a part of nematode present and be functional (as described above), while the roundworm in the inventive methods / assays is used, i. while the nematode contacted with the compounds to be tested becomes.

• – mindestens einer Muskelzelle oder einem Muskelgewebe, und vorzugsweise in dem (mindestens einer Zelle des) Pharynxmuskel(s), Körperwandmuskel(s) und/oder Vulvamuskel(s);
• – mindestens einem Neuron/einer Nervenzelle, und vorzugsweise in (mindestens einem der) M4-Neuronen; und/oder
• – mindestens (einer Zelle des) Gastrointestinaltraktes (d.h. dessen Wand), wie beispielsweise dem Darm (der Darmwand).

As already mentioned above, the Kv4 channel may be expressed in the entire roundworm under these suitable conditions, or only in one or more specific part (s), organ (s), tissue (s) or cell (s) thereof. Preferably, the Kv4 channel is (at least) expressed according to the invention in:

• - At least one muscle cell or muscle tissue, and preferably in the (at least one cell of the) pharyngeal muscle (s), body wall muscle (s) and / or Vulvamuskel (s);
• At least one neuron / nerve cell, and preferably in (at least one of) the M4 neuro NEN; and or
• - At least (one cell of) the gastrointestinal tract (ie its wall), such as the intestine (the intestinal wall).

Even though the invention in its broadest sense is not limited thereto leads the as described herein Kv4 channel expression in the roundworm preferably to a "detectable phenotypic change "(as above described) in the nematode, i. compared to either Wild-type / N2 and / or compared to the original "background" (i.e., stem or line), which for expression of the Kv4 channel was used, and with a suitable detection method is determined; Some non-limiting examples of this are below specified. If the detectable phenotypic change using a appropriate detection method is determined, the change generally at least 1%, preferably 5%, and possibly up to 10% and more of the measured value (s) in comparison to the corresponding value (s) measured in wild type / N2 and / or the original one Background.

The detectable phenotypic change is preferably a detectable biological change, and in particular at least a phenotypic, physiological, behavioral and / or biochemical change in the roundworm, for example as generally in the above mentioned Applications of the applicant described.

Especially preferred is the at least one detectable phenotypic change connected with - and usually the result of - the biological activity of the Kv4 channel in the nematode, i. in the cell (s), part (s), Tissue (s) or organ (s) in which the Kv4 channel is expressed.

Becomes the Kv4 channel in, for example, the muscle and / or neurons is the at least one detectable phenotypic change preferably a change, those with the function of at least one muscle or muscle tissue and / or with the function of at least one neuron or type of Neurons in the worm is connected. If the Kv4 channel is expressed in the gut, is the at least one detectable phenotypic change preferably a change, which is connected to the function of the digestive tract of nematode worm is.

In a preferred embodiment the detectable phenotypic change is a detectable change one or more [of the following functions]: exercise, pharynx pumps (for example, expressed as number of pharyngeal contractions or "pumping movements" per unit time), drinking (e.g., measured with an assay involving the uptake of a fluorescent dye measures, as described in applicant's PCT / IB00 / 00575), oviposition, Hatch of eggs, defecation, Mating behavior, starvation, mortality. Some other, non-limiting biological changes include a response to external stimuli, such as mechanical and / or chemical stimuli (e.g., chemotaxis); an answer to food occurrences; a Reply to (changes the) temperature; an answer to exposure to certain chemical substances; Beginning and / or end of the duration stage; amendments of growth; amendments the life cycle; amendments of metabolism; amendments the neuronal function; and / or changes in muscle function (e.g., contraction and relaxation), apparent from e.g. EPG and / or IER. Methods for defecting and / or measuring such detectable phenotypic change (s) are known in the art; it will turn to the above C. elegans standard manuals Referenced. Also the applications described above Applicants generally describe automated methods high throughput for measuring the aforementioned preferred detectable phenotypic changes.

Especially is the detectable phenotypic change resulting from the expression of the Kv4 channel a change of neural function and / or muscle function, including, but not limited on a change the membrane polarization and / or repolarization (see e.g. EPG and / or IER); neural discharge; muscle contraction and / or relaxation; pharynx; Drink; Move; Defecation and / or Egg laying, with a change drinking, exercise and / or oviposition are particularly preferred is because these are easily automated and medium to high throughput can be determined.

In a preferred aspect, the detectable phenotypic change may also represent a change in the occurrence of membrane repolarization, eg from EPG and / or IER, in particular an increase in the repolarization peak (R) as observed in the EPG and / or a change, and especially one Decrease, the time between the excursion peak (E) and the repolarization peak (R), compared to the background used to express the Kv4 channel. As before As already mentioned, for example, the EPGs of exp-2 mutants show a repolarization peak which is reduced or even essentially absent in comparison to the wild type of N2. Expression of a Kv4 channel on an exp-2 background may result in partial or complete restoration of this peak (see 1A to 1C ).

To another preferred but non-limiting embodiment the invention increases the Expression of the Kv4 channel the rate of pharyngeal pumping, in particular in a (mutant) background, which compared to the wild type / N2 having a reduced pharynx pumping. In this non-limiting embodiment For example, the expression of the Kv4 channel may be the rate of pharyngeal pumping increase in comparison to the (mutant) background, but at a rate that is still lower compared to wildtype / N2.

To another preferred but non-limiting embodiment the invention increases the Expression of the Kv4 channel drinking (measured for example by Inclusion of a fluorescent marker such as Calcein AM), in particular in a (mutant) background, which compared to the wild type / N2 has a reduced drinking. In this non-limiting embodiment For example, the expression of the Kv4 channel may be drinking in the Increase comparison to the (mutant) background, but to a level that compared to the wild type / N2 is still reduced.

The Table 1 below shows a list of some more preferred, but non-limiting Combinations of (1) cells / tissues in which the Kv4 channels (the these coding nucleotide sequences) can be expressed; (2) Promoters for use in this expression; (3) C. elegans strains which as starting lines or strains ("Background") can; and (4) the detectable phenotypic change that is a consequence of Expression of the Kv4 channel in the cells / tissues may be.

• – Stamm UG1598, hinterlegt am 25. April 2002, unter der Zugangsnummer LMBP 5851CB; dies ist ein pha-1(e2123ts)III-Stamm, der mit einem humanen Kv4.3-Kanal unter Kontrolle eines myo-2-Promotors transformiert ist;
• – Stamm UG1611, hinterlegt am 25. April 2002, unter der Zugangsnummer LMBP 5852CB; dies ist ein exp-2(sa26ad1426)V-Stamm, der mit einem humanen Kv4.3-Kanal unter Kontrolle eines myo-2-Promotors transformiert ist.

The following C. elegans lines / strains expressing human Kv4.3 were obtained from the Belgian Coordinated Collections of Microorganisms ("BCCM") LMBP Collection, Ghent University, KL Ledeganckstraat 35, B-9000 Gent, in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms:

• - strain UG1598, deposited on April 25, 2002, under accession number LMBP 5851CB; this is a pha-1 (e2123ts) III strain transformed with a human Kv4.3 channel under the control of a myo-2 promoter;
• - strain UG1611, deposited on April 25, 2002, under accession number LMBP 5852CB; this is an exp-2 (sa26ad1426) V strain transformed with a human Kv4.3 channel under the control of a myo-2 promoter.

TABLE 1

The as generated above, expressing the Kv4 channel transgenic nematodes can be used to identify compounds which with "interact" with the Kv4 channel, and / or determine whether a (given or specific) connection "interacts" with the Kv4 channel.

In the context of the present disclosure, "interacting" with a connection means a protein to be tested, such as the Kv4 channel, in general, that this compound influences, changes, modifies and / or otherwise adversely affects at least one property, characteristic and / or activity of the protein, in particular at least one biological property , a characteristic and / or activity of this protein.

To For example, the compound may contact the one to be examined To bind protein, to ligate, to form a complex, one or more multiple hydrogen bonds form, and / or otherwise associate with it, i. so that at least one (biological) property, one characteristic and / or an activity of the protein, changed, modified or otherwise impaired. For example the compound may be close to or close to the protein to be tested of the active site or associate with it (i.e. with a protein-substrate interaction, that is, competitive with the substrate around the binding), the protein at or near the active site is complete or partially block, to another (binding) site of the protein bind allosteric protein, act as cofactor or inhibit the binding of a cofactar, the association of the Promote protein with other proteins or inhibit, etc .; nevertheless, the invention is in its widest Sense not particularly limited to the way in which the connection interacts / associates with the protein.

Especially can in an ion channel, such as the subject Kv4 channels, the Connect (s) the channel completely or partially block or open, the amount of ions that during through a certain period of time through the channel (can), increase or Reduce; and / or opening and / or Shut down influence the activity / activation and / or change the sensitivity change the channel. This interference can be competitive with the native substrate (s) or non-competitive, it may be temporary or permanent, and / or it may be reversible or irreversible (being transient and reversibly, as a rule, is preferred).

Accordingly can (can) the compound (s) act as agonists of the Kv4 channel, as an antagonist of the Kv4 channel and / or as (reversible and / or irreversible) Inhibitor of the Kv4 channel, although the invention is in its widest Senses not limited thereto is. For example, the connection can potentiate the Kv4 channel sensitize or desensitize, or otherwise activity and / or sensitivity of the Kv4 channel or diminish.

Preferably is the above "interaction" with the Kv4 channel such that they are measured by suitable detection methods or can be observed in another way (i.e., qualitatively and / or quantitative), for example using Xenopus oocytes and / or using EPG and / or IER in C. elegans.

Especially preferred - there According to the invention, the interaction the compound (s) with the protein in vivo using the Nematode C. elegans is determined - is the interaction such that they also become a detectable change in the one used Roundworm (roundworm) leads. In particular, the detectable change (referred to herein as "significant biological change") is a detectable one biological change in the threadworm, e.g. at least one phenotypic, physiological, behavioral and / or biochemical change in the threadworm.

Becomes the significant biological change measured using a suitable detection method, is the change generally at least 1%, preferably at least 5%, and possibly to to 10% and more of the (measured) value) compared to the the corresponding value (s) measured on the strain (without being exposed to the compounds).

Especially preferred is the at least one significant biological change in the nematode associated with - and is usually a Result of - the change (s) the activity, sensitivity etc. of the Kv4 channel caused by the connection (s).

is the Kv4 channel in, for example, the muscle and / or neurons is the at least one significant biological change preferably a change, those with the function of at least one muscle or muscle tissue and / or with the function of at least one neuron or type of neurons in the worm. Will the Kv4 channel in the intestine is the at least one significant biological change preferably a change, which is connected to the function of the digestive tract of nematode worm is.

It should be noted here that, in general, the "significant biological change", which depends on the (s) to be tested compound (s) is or may not affect the same property (ie, substantially the same phenotypic, physiological, behavioral and / or biochemical property) as the "detectable phenotypic change" resulting from the expression of the Kv4 channel in the roundworm be acts. For example, in a preferred embodiment, the "detectable phenotypic change" may be a defect in the roundworm (such as a pharyngeal pump, movement, or oviposition defect), and the significant biological change caused by the compound may be the full or partial be partial "recovery" of this defect.

In a preferred embodiment the significant biological change is a detectable change in one or more properties, such as exercise, pharyngeal pumps, (e.g., measured as the number of pharyngeal contractions or "pumping movements" per unit of time), Drinking (e.g., as measured by an assay involving the uptake of a fluorescent dye, as in applicant's PCT / IB00 / 00575 described), oviposition, hatching of eggs, defecation, Mating behavior, starvation, mortality. Some non-limiting biological changes can include a response to external stimuli, such as mechanical stimuli and / or chemical stimuli (e.g., chemotaxis); the answer to that Presence of food; the answer to (changes the) temperature; the answer to exposure to certain chemical substances; Beginning and / or end of the duration stage; Changes in growth; amendments the life cycle; amendments of metabolism; amendments the neuronal function; and / or changes in muscle function (e.g., contraction and relaxation), apparent from e.g. EPG and / or IER. Methods for determining and / or changing such detectable phenotypic change (s) have already been mentioned above.

Especially Preferably, the significant biological change is a change of the neural and / or muscle function, including, but not limited on a change membrane polarization and / or repolarization (see e.g. from EPG and / or IER); neural discharge; Muscle contraction and / or relaxation contribute; pharynx; Drink; Move; Defecation and / or Egg laying, with a change drinking, exercise and / or oviposition are particularly preferred is.

In a preferred embodiment, the significant biological change may also be a change in the membrane repolarization, evident eg from EPG and / or IER, in particular an increase of the repolarization peak (R), evident in the EPG, and / or a change, in particular a decrease, of Time between the excitation peak (E) and the repolarization peak (R) compared to the background used to express the Kv4 channel. For example, as noted above, the EPGs of exp-2 mutants show a repolarization peak that is reduced or even substantially absent from N2 in the wild type. Expression of a Kv4 channel on an exp-2 background may result in partial or complete restoration of this peak (see 1A to 1C ).

The Table 2 below shows, for example, a list of some preferred, but not restrictive Combinations of (1) cells / tissues where the Kv4 channels (the these coding nucleotide sequences) can be expressed; (2) Promoters for use in this expression; (3) C. elegans strains which as starting lines or strains ("Background") can; and (4) the relevant significant biological change from the to be examined compound (s) in such nematode causes can be.

TABLE 2

• (a) in-Kontakt-bringen mindestens eines einen Kv4-Kanal exprimierenden Fadenwurms mit (mindestens) einer Verbindung oder Verbindungen; und
• (b) Detektieren und/oder Beobachten mindestens einer signifikanten biologischen Änderung (wie vorstehend definiert) in oder bei dem Fadenwurm.

In general, the method for determining whether a compound or compounds can interact with the Kv4 channel involves the following steps:

• (a) contacting at least one Kv4 channel-expressing roundworm with (at least) one compound or compounds; and
• (b) detecting and / or observing at least one significant biological change (as defined above) in or at the roundworm.

• (a) in-Kontakt-bringen einer oder mehrerer Verbindungen der Gruppe von Verbindungen mit mindestens einem Fadenwurm, wie vorstehend beschrieben, und
• (b) Detektieren und/oder Beobachten, ob eine der Verbindungen der Gruppe eine signifikante biologische Änderung in oder bei dem Fadenwurm bewirkt.

The method for screening a group of compounds for the presence of one or more compounds in the group that can interact with the Kv4 channel generally comprises the following steps:

• (a) contacting one or more compounds of the group of compounds with at least one nematode, as described above, and
• (b) detecting and / or observing whether one of the compounds of the group causes a significant biological change in or at the roundworm.

As mentioned above, in step (b) of both of these methods, that in the nematode worm observed significant change at least indicative - and more preferably representative - for an interaction the connection (s) with the Kv4 channel, in qualitative and preferably also quantitatively. The above steps (a) and (b) will be closer below described.

• – indem die Verbindung(en), wahlweise in einer geeigneten Formulierung, wie einer Lösung oder Suspension, (direkt) mit dem Körper und/oder der Kutikula ("Haut") des Fadenwurms in Kontakt gebracht wird (werden), und/oder indem die Verbindung auf andere Art in die Umgebung des Fadenwurms eingebracht wird, wie beispielsweise durch Aufnahme der Verbindung(en) in das Medium, in oder auf dem der Fadenwurm gehalten, gezüchtet, kultiviert oder erhalten wird (wobei dieses Medium auch in geeigneter Weise die Expression des Kv4-Kanals induzieren kann). Es ist gut bekannt, dass Fadenwürmer unter diesen Umständen Verbindungen aus ihrer Umgebung direkt über ihre Kutikula (z.B. durch Diffusion oder aktiven Transport, manchmal auch als "Einweichen" bezeichnet) und/oder über Öffnungen in ihrer Kutikula, wie die sensorischen Amphidneuronen, aufnehmen können;
• – indem die Verbindung(en) in den Körper des Fadenwurms injiziert oder auf andere Art eingeführt werden, beispielsweise in die Blutbahn oder in einen beliebigen Teil oder Teile des Verdauungstraktes;
• – indem der Fadenwurm mit der (den) Verbindung(en) gefüttert und/oder die Verbindung(en) in einer geeigneten Nahrungsquelle, Futterzusammensetzung oder eine andere geeignete Substanz oder ein Material aufgenommen werden, welches die Würmer über Fressen oder Pharynxpumpen aufnehmen können (z.B. Mikrosphären), und anschließend diese Nahrungsquelle usw. an die Fadenwürmer verfüttert wird.

In step (a), the roundworm (ie at least one cell, part, tissue or organ of the roundworm) is brought into contact with the compound (s) to be tested. This can be done in any suitable way, such as:

• By bringing the compound (s), optionally in a suitable formulation, such as a solution or suspension, into contact (directly) with the body and / or the cuticle ("skin") of the roundworm, and / or by the compound is otherwise introduced into the vicinity of the nematode, such as by uptake of the compound (s) into the medium, in or on which the nematode is kept, bred, cultured or maintained (this medium also being suitable for expression) of the Kv4 channel). It is well known that under these circumstances, nematodes can pick up compounds from their environment directly via their cuticle (eg, by diffusion or active transport, sometimes called "soaking") and / or through openings in their cuticle, such as the sensory amphid neurons ;
• By injecting or otherwise introducing the compound (s) into the body of the roundworm, for example into the bloodstream or into any part or parts of the digestive tract;
• By feeding the roundworm with the compound (s) and / or ingesting the compound (s) in a suitable food source, feed composition or other suitable substance or material which the worms may receive via seizures or pharynx pumps (eg Microspheres), and then this food source, etc. is fed to the nematode worms.

These and other methods to examine the roundworms with the one to be examined To contact compound (s), and appropriate conditions therefor are known in the art. Generally, the invention is not particularly limited in the way in which the nematode with the compound (s) to be investigated is brought into contact as long as the compound (s) of the threadworm absorbed or otherwise absorbed (being), so that the compound in contact with those expressed by the nematode Kv4 channel (Kv4 channels) can come; and preferably so that - if the connection with the the Kv4 channel (s) can interact as described here - contacting the roundworm with the link to a significant biological change leads (as described above).

For example depends on that Time, while which exposed the thread worm to the compound (s) or so is based on factors such as the used nematode (including the specific used Strain), the way in which the nematode worm with the compound (s) the amount of compound (s) with which the nematode is brought into contact (i.e., both the absolute Amount and / or concentration), the significant to be measured biological change, and similar Factors. The concentration and / or the amount of the compound (s), which the threadworm is exposed to depends usually on factors like the nematode used, the way in which the nematode worm is brought into contact with the compound, the significant to be measured biological change, the (intended) exposure time, and similar factors.

in the Generally, one skilled in the art may appropriate time, amount and concentration determine the exposure of the compound, optionally after initial routine trial-and-error experiments in limited Number, and / or by comparison with one or more reference compounds, known to interact with Kv4 channels (see below).

The number of roundworms that is brought into contact with the compound (s) to be tested (ie, the "sample size") is not critical, and may for example depend on the size of the container / vessel used. Usually the sample comprises between 2 and 500, preferably between 3 and 300, and in particular between 5 and 200, very particularly preferred between 10 and 100 Fadenwür mer. When the assay is performed in multi-well plates (as described below), each well will usually contain between 15 and 75 worms, for example, between 20 and 50 worms. Although not preferred, it is not excluded that the sample consists of a single worm. Also, although it is usually not preferred that only a part, tissue, organ or cell of a nematode of the invention be used, such as (isolated) muscle tissue, an isolated pharynx, etc., it is possible.

Even though the invention in its broadest sense is not limited thereto it is ordinary prefers that the thread worms in each sample worms include, which - at least at the beginning of the assay - im Essentially the same, i. E. that they are essentially of from the same strain (e.g., expressing the Kv4 channel); in the Are of isogenic genotype; essentially the same, Show Kv4-related genotype; are substantially "synchronized" (i.e., substantially synchronized) same stage of development, but during the Change the course of the assay can - and usually will change - not for all worms the sample at the same rate and / or in the same way); essentially cultivated / cultivated in the same way were (for example in the same medium).

Further Conditions for contacting the roundworm with the (the) test compound (s) - such as temperature, the medium and the equipment used, etc. - may be suitable for the person skilled in the art chosen again, optionally after initial routine trial-and-error experiments in limited Number, and again based on the considerations given above. For example, you can these conditions - however not compulsory - general the same as the conditions for cultivating / breeding the Nematodes, as described above (and therefore may be such that an expression of the Kv4 channel is induced / obtained). However, these conditions can the use of temperatures, media, buffers and reagents specifically for exploring / screening compounds specifically are suitable, e.g. as in the applicant's aforementioned applications described. Further, though usually not preferred, further includes the invention also includes the exposure of nematode before or simultaneously with exposure to the compound (s) to be tested, with one or more compounds containing at least one native Process or activity increase or decrease the threadworm, for example, a native present ion channel. The conditions used should however for the used threadworms at least physiologically acceptable, at least during the Time, while the methods of the invention carried out become.

• – eine Kontaktzeit zwischen 1 Minute und 24 Stunden, insbesondere zwischen 10 Minuten und 12 Stunden, besonders bevorzugt zwischen 0,5 und 8 Stunden, wie beispielsweise zwischen 1 und 6 Stunden, z.B. etwa 2, 3, 4 oder 5 Stunden;
• – eine Konzentration/Menge der Verbindung(en) zwischen 0,01 und 1.000 mM (abhängig von der Toxizität und Löslichkeit der untersuchten Verbindung), insbesondere zwischen 1 und 500 mM, besonders bevorzugt zwischen 5 und 300 mM, wie beispielsweise zwischen 5 und 100 mM, z.B. etwa 10, 20, 30, 40, 50, 60, 70, 80 oder 90 mM; wobei die Verbindung(en) wahlweise mit einer geeigneten Futterquelle, wie beispielsweise E. coli (in den vorstehend angegebenen Mengen verwendet) (vor-)gemischt sein kann (können);
• – einer Temperatur zwischen 15 und 30°C, insbesondere zwischen 17 und 25°C, wie beispielsweise etwa 18, 19, 20, 21, 22, 23 oder 24°C;
• – die Verwendung eines geeigneten flüssigen, viskosen oder halbviskosen Mediums, wie beispielsweise Agar, M9, S-Puffer, und/oder eines der vorstehend angegebenen Medien; auf einer Platte, in einer Petrischale, in einem Röhrchen, in der Vertiefung einer Multi-Well-Platte, oder in einem anderen geeigneten Gefäß oder Behälter;
• – eine Probengröße zwischen 10 und 1.000 Fadenwürmern, insbesondere zwischen 25 und 200 Fadenwürmern, besonders bevorzugt zwischen 75 und 150 Fadenwürmern.

Some suitable, non-limiting conditions may be, for example:

• A contact time between 1 minute and 24 hours, in particular between 10 minutes and 12 hours, more preferably between 0.5 and 8 hours, for example between 1 and 6 hours, for example about 2, 3, 4 or 5 hours;
• A concentration / amount of the compound (s) between 0.01 and 1000 mM (depending on the toxicity and solubility of the tested compound), in particular between 1 and 500 mM, more preferably between 5 and 300 mM, for example between 5 and 100 mM, eg about 10, 20, 30, 40, 50, 60, 70, 80 or 90 mM; wherein the compound (s) may optionally be (are) pre-mixed with a suitable food source, such as E. coli (used in the amounts indicated above);
• A temperature between 15 and 30 ° C, especially between 17 and 25 ° C, such as about 18, 19, 20, 21, 22, 23 or 24 ° C;
• The use of a suitable liquid, viscous or semi-viscous medium, such as agar, M9, S buffer, and / or one of the media indicated above; on a plate, in a petri dish, in a tube, in the well of a multi-well plate, or in another suitable vessel or container;
• - A sample size between 10 and 1000 roundworms, in particular between 25 and 200 roundworms, more preferably between 75 and 150 roundworms.

Further suitable conditions for examining / screening compounds using nematodes are in the aforementioned Registrations of the applicant indicated. Is the significant to be measured biological change for example, exercise, pharyngeal pumps, oviposition or defecation, are some particularly suitable conditions for assays in the international Application PCT / IB00 / 00575 (WO 00/63427).

Once the roundworms have been exposed to the compound (s) to be tested, the significant biological change caused by the exposure may be measured / observed in any suitable manner visually or non-visually, as the case may be determining phenotypic, physiological, behavioral and / or biochemical property (s). Suitable methods are known to the skilled person; reference is made to the aforementioned C. elegans manuals and applicant's applications and to the description below.

amendments of neural function For example, be determined by means of EPG and / or IFR. amendments in the rate of pharyngeal pumping can visually by counting the number of pharyngeal contractions ("pumping movements") the time can be determined with a microscope and a stopwatch. amendments in the drink can be measured by recording a fluorescent marker, such as Calcein-AM, over the time is determined as described in International Application PCT / IB00 / 00575 (WO 00/63427) of the applicant; there are also methods for the determination of movement, oviposition and defecation.

The thus measured / determined significant biological changes indicate the interaction of the compound with the Kv4 channel, and are preferably a measure of, and allow therefore, the identification of compound (s) associated with the Kv4 channel interact, or determining the interaction of a compound with the Kv4 channel.

According to the following may Compound (s) to be investigated: in a preferred, but non-limiting embodiment these are "small ones Molecules ", hereby is general a molecular unit having a molecular weight of less than 1,500 meant, preferably less than 1,000. This can be, for example be an organic, inorganic or organometallic molecule, which may also be in the form of a suitable salt, such as a water-soluble Salt; it can also be called complex chelated and / or similar Molecular unit, as long as the (total) molecular weight within the aforementioned range.

In a preferred embodiment will such a "small Molecule "in agreement with and / or according to the criteria of at least one, preferably at least two, more preferably at least three, up to to all of the so-called Lipinski rules for predicting the drug likelihood (see Lipinski et al., Advanced Drug Delivery Reviews 23 (1997), pages 3-25). As is known in the field, are small molecules which meet these criteria, particularly suitable (as starting points) for construction and / or Development of medicines for human use, and can be used as starting points, for example serve in "hits-to-leads" chemistry, and / or as starting points for the lead development (where the inventive method also applied can be).

For this Purposes considered the construction of small molecules (as well as the construction libraries that consist of such small molecules) also the presence of pharmacophore points, for example according to the methods Muegge et al., J.Med.Chem. 44, 12 (2001), pp. 1-6, and the documents cited therein.

Of the Expression "small Peptide " generally (oligo) peptides that are between 2 and 35, for example between 3 and 25 amino acids include (e.g., in one or more linked chains, and preferably in a single chain). It can be seen that some of these small peptides also from the term small molecule, like him used herein are, depending on their molecular weight.

In a preferred, but non-limiting embodiment For example, the invention is used to construct a group or library of (related or otherwise unrelated) small molecules, for example a standard "robustness set", a primary one Screening library (for example, unrelated otherwise Connections), a combined library, a number of closely related chemical analogs and / or a group of known blockers and / or openers for other indoor channels (like a group of known blockers or openers for other (voltage dependent) potassium channels); etc. to screen. Such groups or libraries are those skilled in the art can and can be seen For example, but not limited to, commercially available chemical libraries, such as the various of Tocris Cookson, Bristol, GB available Libraries include.

It will be appreciated by those skilled in the art that for the screening of groups and / or libraries of compounds, it is often preferred to carry out the method of the invention at medium to high throughput. For this, the inventive methods are preferably carried out automatically (ie using suitable machines), and / or in a suitable multi-well plate format, such as standard plates with 6, 24, 48, 96, 384, 1536, or 3072 wells (each well being a Multi-well plate may contain a separate sample of worms). Reference is made, inter alia, to the above-mentioned applications of the Applicant - and more particularly International Application PCT / IB00 / 00575 (WO 00/63427) - describing not only automated, medium to high throughput methods for cultivating, maintaining and screening roundworms in multi-well plates - but also automated, non-visual methods for measuring (changes in) phenotypic, physiological, behavioral and / or or biochemical properties of roundworms (such as drinking, agitation, defecation and oviposition) which may be used in accordance with the invention as a "significant biological change".

In the inventive method each individual sample of roundworms is generally a single one exposed to compound under study at a single concentration; where different samples are either two or more different be exposed to compounds under investigation; two different ones Concentrations of the same compound (e.g., a dose-response curve for this Make a connection), or a combination of them.

The Invention also includes that (a sample of) nematode worm (s) two or more connections are (are) exposed to - in the Essentially the same time or sequentially (e.g., with an intermediary Washing step) - to For example, to determine whether the two compounds the same or a different effect to the two individual compounds (for example, whether they provide a synergistic effect, or an inhibitory or competitive effect). Farther can the methods of the invention include the use of one or more reference samples, e.g. Samples without the presence of any compound (s), and / or in the presence of a certain amount of a known reference compound. The frame according to the invention also includes the use of two or more samples of roundworms of different strains (such as from two different strains, the Kv4 channels express, and / or reference strains, which do not express Kv4 channel as mentioned above), e.g. the effect of the compound (s) being tested on these different ones strains to compare.

Out It will be apparent to those skilled in the art above that the above usually described procedures by means of an assay or a screen, and these assays and screens are further aspects of the invention.

For purposes In the present disclosure, "agonists" of the Kv4 channel are broadly defined as compounds that control the flow of potassium ions through the Kv4 channel increase, while "antagonists" of the Kv4 channel as Compounds are defined which control the flow of potassium ions through reduce the Kv4 channel. In this regard, it should be noted that "blocker" of the Kv4 channel as an agonist and / or as an antagonist of the Kv4 channel (mainly depending on whether the connection is opening or closing the Kv4 channel), while "openers" of the Kv4 channel are usually agonists.

For example can Agonists and antagonists in general the following non-limiting (s) significant biological change (s) on hKv4-expressing strains provide and thereby be identified:

TABLE 3

Just for illustration, using the methods of the invention for example, found that 4-aminopyridine, a compound that acts as a general antagonist of (voltage-dependent) potassium channels is known, the following significant biological changes in the following hKv4.3-expressing roundworms (in the specified concentrations and contact times):

TABLE 4

• – Herzkrankheiten, wie beispielsweise: Arrhythmie, Tachykardie und kongestives Herzversagen;
• – Krankheiten des Nervensystems, wie beispielsweise: Epilepsie, Schlaganfall, Hirntrauma, Angstzustände, Schlaflosigkeit, Alzheimer'sche Krankheit und Parkinson-Syndrom.

In the field of pharmacy and / or veterinary medicine, the invention may be used to identify and develop compounds useful in the following disease fields (ie, in the preparation of preparations for the prevention, treatment and / or curing of the indicated diseases /Conditions):

• - heart disease, such as: arrhythmia, tachycardia and congestive heart failure;
• - Diseases of the nervous system, such as: epilepsy, stroke, brain trauma, anxiety, insomnia, Alzheimer's disease and Parkinson's syndrome.

Further Disease areas where the compounds may be useful are Hypertension and urinary incontinence.

It is expected for a pharmaceutical application opener of the Kv4 channel identified / developed using the invention will be of particular importance.

For a pharmaceutical Application can the compounds as the free acid or base, and / or as a pharmaceutically acceptable acid addition and / or base addition salt (e.g., obtained with non-toxic organic or inorganic acid or base), as a hydrate, Solvate and / or complex, and / or as a prodrug, for example as an ester. Such salts, hydrates, solvates, etc. and their preparation are known in the art; For example, it is referred to in the Salts described in US-A-6,372,778, US-A-6,369,086 and US-6,369,067, Hydrates, solvates, etc.

in the Generally can for one pharmaceutical use the compounds as a pharmaceutical preparation, containing at least one compound of the invention and at least a pharmaceutically acceptable carrier, solvent or excipient and / or an adjuvant, and optionally one or more further pharmaceutically active compounds. As non-limiting examples such formulation may be suitably administered for oral administration, for parenteral administration (such as intravenous, intramuscular or subcutaneous injection or as intravenous infusion), for topical Administration, for administration as an inhalation, as a skin patch, as an implant, as a suppository etc. are available. Suitable administration forms - depending on the mode of administration can these have a solid, semi-solid or liquid form - as well Method and carrier for use in their manufacture are known in the art; again, for example, US-A-6,372,778, US-A-6,369,086 and US 6,369,067.

The Pharmaceutical preparations are preferably in unit dosage form before, and may be suitable be packaged, for example in a box, a blister, a vial, a bottle, a bag, an ampoule or in any other suitable container or container (which can be labeled accordingly) optionally with one or more leaflets, contain the product information and / or instructions for use. In general, these dosage units comprise between 1 and 500 mg of at least one compound of the invention, e.g. about 10, 25, 50, 100, 200, 500 or 1,000 mg per dose unit.

to Pharmaceutical use is generally at least one compound in an amount between 0.01 and 150 mg / kg of body weight per day to the patient administered, distributed over one or more doses per day. The quantity (s) to be administered and the further treatment regimen will be provided by the attending physician determined, depending of factors such as age, gender and general condition of the patient and the nature and severity of the disease / symptoms to be treated.

On In the field of agrochemicals, the invention can be used to Identify compounds known as pesticides, insecticides, Nematozide and / or biocides or as pesticides are suitable. For example, you can the compounds to combat of the species listed in US-A-6,372,774. Therefor can the compounds (or a suitable salt, hydrate or ester thereof) suitably formulated with one or more agrochemically acceptable carriers be an appropriate formulation for an agrochemical application as is known to those skilled in the art (for example to the formulations and uses described in US-A-6,372,774 referenced).

The Compounds and compositions may also be used in the field of Veterinary medicine can be used either to treat diseases (such as the above) in animals (e.g., mammals) to treat, and / or attack by parasites or others harmful Organisms in animals (e.g., mammals) or to prevent. Formulations of compounds of the invention suitable for these Aims are also known to those skilled in the art for example, see US-A-6,372,774).

The Invention will now be described by the following non-limiting experimental part and the non-limiting figures, wherein:

1 shows an electropharyngogram of C. elegans strain UG 1547 expressing human Kv4.3 under the control of a myo-2 promoter (on an N2 background);

2 and 3 Photographs which show the expression of the human Kv4.3 channel in the pharyngeal muscle of the strain UG 1598 (Example III) or of the strain UG 1611 (Example IV). The photographs were taken after immunohistochemical staining with an anti-human Kv4.3 rabbit antibody as described in Examples III and IV;

4 Figure 4 is a graph of the number of pharyngeal pumping movements per 30 second interval, with and without the presence of 4-aminopyridine in strain UG 1598 (according to the invention) and in an N2 reference strain (comparison);

5 Figure 4 is a graph of the number of pharyngeal pumping movements per 30 second interval, with and without the presence of 4-aminopyridine in the strain UG 1611 (according to the invention) and in an exp-2 (sa26as1426) V background (comparison);

6 schematically shows the expression of the vector pDW 2700 used in Example II;

7 a graph showing the fluorescent marker Calcein AM in the drinking assay from PCT / IB00 / 00575 by N2 (reference) and the strain UG 1598 (according to the invention), wherein the uptake of the fluorescent marker calcein AM by N2 is rated as 100% ;

8th a graph for the uptake of the fluorescent marker calcein AM in the drinking assay from PCT / IB00 / 00575 by N2 and exp-2 strains (reference) and the strain UG 1611 (according to the invention) shows, wherein the uptake of the fluorescent marker calcein AM by the exp-2 background rated as 100%;

9 a graph showing schematically the influence on the uptake of the fluorescent marker calcein AM in the drinking assay from the PCT / IB00 / 00575 by the exp-2 strain (reference) and the strain UG 1611 (according to the invention), wherein the uptake of the fluorescent Markers calcein AM is valued by the exp-2 background without the presence of 4-aminopyridine as 100.

EXPERIMENTAL PART

A full WT hKv4.3 coding cDNA was isolated from a human cDNA library Brain cloned. Unless otherwise stated, all cloning steps were used performed using standard methods, i. as from the manufacturers provided reagents / test kits, and / or how described in Ausubel et al. and / or Sambrook et al., supra. An anti-human Kv4.3 rabbit antibody was P 0358, derived from Sigma (St. Louis, Missouri, USA). An anti-rabbit IgG Cy3 conjugate was C 2306, also purchased from Sigma (St. Louis, Missouri, USA).

Example I:

Cloning a WT hKv4.3 complete Length coding cDNA

In a 20 ul reaction, a PCR was performed using Advantage ^® -GC 2 PCR (Clontech, Palo Alto, CA, USA) according to the manufacturer's performed with the specific primer combination OGV 1 / oGV4 and 1, ul Marathon -Ready ^™ human brain cDNA (Clontech). The PCR conditions were as follows: initial denaturation at 95 ° C for 1 minute, followed by 20 cycles of PCR (15 "at 95 ° C, 3 'at 68 ° C) followed by 20 cycles of PCR (95 ° C for 15 ", at 68 ° C for 3 '(+5" / cyclr)).

The obtained PCR products were analyzed by agarose gel electrophoresis analyzed and interesting DNA was isolated and with TA clones cloned into the pCR-XL-TOPO vector (Invitrogen). After provision of appropriate Restriction sites were constructed a full-length cDNA of the WT hKv4.3 cDNA (see SEQ ID NO: 4, wherein the corresponding amino acid sequence shown in SEQ ID NO: 5).

The resulting plasmid was designated pGV7 (using the primer combination oGV1 [SEQ ID NO: 1] / oGV4 [SEQ ID NO: 2]).
oGV1: AGGGGTTTGCTGAACTAACTCCAAGCTGG
oGV4: TTCATTCCCCACTACCCRCTCTGGCCCTCTGTCC

Example II:

Cloning of the WT hKv4.3 cDNA in the C. elegans Myo-2 expression vector

A 2172 bp Spe1 / Xba1 fragment of pGV7 was isolated, purified and inserted into the Nhe1 cloning site of the expression vector pDW2700 (shown schematically in FIG 6 ) to bring hKv4.3 under the control of the myo-2 promoter present in the vector.

The obtained plasmid was designated pGV8.

Example III: Transformation a C. elegans pha-1 mutant with a human Kv4.3 coding sequence

Of the C. elegans strain pha-1 (e2123ts) ITI, obtained from CGC (CGC strain GE24) was mixed with a mixture containing 5 ng / μl plasmid pGV8 (Example II), 20 ng / μl GFP marker (pDW2821) and 5 ng / μl pBX rescue fragment of the pha-1 phenotype, added with genomic C. elegans DNA (Sau3A digested), injected into the gonad, to form a complex arrangement (Kelly, Xu and Fire, Worm Breeders Gazette 14 (1): 64 (1 October 1995)). The injection was performed according to the in I. A. Hope, supra, described standard protocol.

The thus obtained F1 threadworms were successful on transformation with the pGV8 plasmid and the expression of the hKv4.3 channel using the GFP marker selected. The hKv4.3 expressing pha-1 strain thus obtained was used as a strain UG1598 and was on 25 April 2002 at the BCCM under the Access number LMBP 5851 CB deposited.

The expression of the hKv4.3 channel in the pharyngeal muscle was confirmed by fluorescent immunohistochemistry using the whole mount method essentially as described in Steinbusch et al., Acta Histochem S35, 85-106 (1998). human Kv4.3 rabbit antibody P 0358 (1/10 dilution) was used as the primary antibody and anti-rabbit IgG Cy3 conjugate C2306 (1/1000 dilution) as the secondary antibody (see 2 ). It appears that the expressed Kv4.3 channel is localized primarily, but not exclusively, in the cell membrane, thus corresponding to the expected localization for a Kv4.3-like channel.

Example IV: Transformation a C. elegans exp-2 mutant expressing a human Kv4.3 sequence

Of the C. elegang's strain exp-2 (sa26ad1426) V, obtained from CGC (CGC strain DA 1426) was added to a mixture containing 0.1 ng / μl of plasmid pGV8 (Example II), 20 ng / μl GFP marker (pDW2821), phenotype, added with genomic C. elegans DNA (Sau3A digested), injected into the gonad, to form a complex arrangement (Kelly, Xu and Fire, Worm Breeders Gazette 14 (1): 64 (1 October 1995)). The injection was performed according to the in I. A. Hope, supra, described standard protocol.

The thus obtained F1 threadworms were successful on transformation with the pGV8 plasmid and the expression of the hKv4.3 channel using the GFP marker selected. The hKv4.3 expressing exp-2 strain thus obtained was used as a strain UG1611 and was on April 25, 2002 at the BCCM under the Access number LMBP 5852CB deposited.

Expression of the hKv4.3 channel in the pharyngeal muscle was confirmed by fluorescent immunohistochemistry using the whole mount method, essentially as described in Steinbusch et al., Acta Histochem S35; 85-106 (1998), where anti-human Kv4.3 rabbit antibody P 0358 (1/10 dilution) was used as the primary antibody and anti-rabbit IgG Cy3 conjugate C2306 (1/1000 dilution) as the secondary antibody (see 3 ).

Example V: Influence of hKv4.3 expression on pharnyx pumping in a C. elegans pha-1 tribe

Of the hKv4.3 expressing C. elegans strain UG 1598 (Example III) in terms of the rate of pharyngeal pumping. As a comparison served N2 background.

The nematodes were cultured on agar plates until the L4 stage, and then overnight 20 ° C to to reach the adult stage. The number of pharyngeal "pumping movements" (contractions) each 30 seconds was determined visually using a stopwatch.

The results are in 4 shown schematically. It can be seen that strain UG 1598 shows a greatly reduced rate of pharyngeal pumping compared to the background.

Example VI: Influence of 4-aminopyridine on the pharynx pump in an hKv4.3 expressing pha-1 strain

Of the Influence of 4-aminopyridine, a known general antagonist of the voltage-dependent Potassium channel, at the rate of pharyngeal pumping at a hKv4.3 expressing C. elegans strain UG 1598 (Example III) was determined as follows.

The nematodes were cultured on agar plates until the L4 stage, and then overnight 20 ° C to to reach the adult stage. The worms were subsequently on 0.5 mM 4-aminopyridine in DMSO containing agar plates transferred.

To 3 hours at 20 ° C was the number of pharyngeal "pumping movements" (contractions) each 30 seconds visually determined using a stopwatch. As a comparison, the influence of 4-aminopyridine on an N2 background was shown in principle determined in the same way.

The results are in 4 shown schematically. It can be seen that 0.5 mM 4-aminopyridine increases the rate of pharynx pumping in strain UG 1598, but has a very small influence on the rate of pharyngeal pumping in the background.

Example VII: Influence of hKv4.3 expression on pharyngeal pumping in a C. elegans exp-2 strain

Of the hKv4.3 expressing C. elegans strain UG 1611 (Example V) in terms of the rate of pharyngeal pumping. As a comparison served original exp-2 background.

The nematodes were cultured on agar plates until the L4 stage, and then overnight 20 ° C to to reach the adult stage. The number of pharyngeal "pumping movements" (contractions) each 30 seconds was determined visually using a stopwatch.

The results are in 5 shown schematically. It can be seen that the strain UG 1611 shows a slightly increased rate of pharyngeal pumping (ie, "restoration" of the exp-2 phenotype) compared to the exp-2 background.

Example VIII: Influence of 4-aminopyridine on the pharynx pump in an hKv4.3 expressing exp-2 strain

Of the Influence of 4-aminopyridine, a known general antagonist of the voltage-dependent Potassium channel, at the rate of pharyngeal pumping at a hKv4.3 expressing C. elegans strain UG 1611 (Example IV) was determined as follows.

The nematodes were cultured on agar plates until the L4 stage, and then overnight 20 ° C to to reach the adult stage. The worms were subsequently on 2 mM 4-aminopyridine in DMSO containing agar plates transferred. To 3 hours at 20 ° C was the number of pharyngeal "pumping movements" (contractions) each 30 seconds visually determined using a stopwatch. As a comparison, the influence of 4-aminopyridine on an original exp-2 background in principle determined in the same way.

The results are in 5 shown schematically. It can be seen that 2 mM 4-aminopyridine reduces the rate of pharyngeal pumping of strain UG 1611 but has a very small impact on the rate of pharyngeal pumping at the exp 2 background.

Example IX: Influence of hKv4.3 expression on drinking behavior in a C. elegans pha-1 strain

Of the hKv4.3 expressing C. elegans strain UG 1598 (Example III) in terms of of drinking behavior by recording over the Time of the fluorescent dye calcein AM, essentially as described in Examples 2 and 3 of the international application PCT / IB00 / 00575 of the applicant, using a plate with 96 wells containing 100 roundworms / well. When Comparison served N2.

The results are in 7 schematically, wherein the uptake of calcein AM by the N2 strain was rated at 100%. It can be seen that the strain UG 1598 shows a reduced drinking compared to N2 (ie 76% for UG 1598 compared to 100% for N2).

Example X: Influence of hKv4.3 expression on drinking behavior in a C. elegans exp-2 tribe

Of the hKv4.3 expressing C. elegans strain UG 1611 (Example IV) in terms of of drinking behavior by recording over the Time of the fluorescent dye calcein AM, essentially as described in Examples 2 and 3 of the international application PCT / IB00 / 00575 of the applicant, using a plate with 96 wells containing 100 roundworms / well. When Comparison was the exp-2 (sa26as1426) V background.

The results are in 8th schematically, wherein the uptake of calcein AM by the exp-2 strain was rated at 100%. It can be seen that the exp-2 background shows reduced drinking compared to N2 (ie 100% at exp-2 compared to 156% at N2). In the strain UG 1611, the expression of the hKv4.3 channel in the exp-2 background increases the uptake of the marker to 114%.

Example XI: Influence of 4-aminopyridine on drinking behavior in an hKv4.3 expressing exp-2 strain

Of the Influence of 4-aminopyridine, a known general antagonist of the voltage-dependent Potassium channel, on the drinking behavior in the hKv4.3 expressing C. elegans strain UG 1611 (Example IV) was determined by measuring uptake of the fluorescent dye calcein AM with and without presence of 0.3 mM 4-aminopyridine, essentially as described in Examples 2 and 3 of the international application PCT / IB00 / 00575 of the applicant.

The results are in 9 schematically, wherein the uptake of calcein AM by the exp-2 strain without the presence of 4-aminopyridine was rated at 100%. It can be seen that in the strain UG 1611 0.3 mM 4-aminopyridine reduces the uptake of the fluorescent marker in this drinking assay from 114% to 109%; whereas in the exp-2 background, 0.3 mM 4-aminopyridine increases the uptake of the fluorescent marker from 100 to 103% in this drinking assay.

Example XII: Influence hKv4.3 expression on the electropharyngogram in C. elegans N2

Of the C. elegans strain N2 was mixed with a mixture containing 5 ng / μl of plasmid pGVB (Example II), 20 ng / μl GFP marker (pDW2821) and 5 ng / μl pBX rescue fragment of the pha-1 phenotype, added with genomic C. elegans DNA (Sau3A digested), injected into the gonad, to form a complex arrangement (Kelly, Xu and Fire, Worm Breeders Gazette 14 (1): 64 (1 October 1995)). The injection was performed according to the in I. A. Hope, supra, described standard protocol.

The thus obtained F1 threadworms were successful in transforming with the pGVB plasmid and the expression of the hKv4.3 channel using the GFP marker selected. The hKv4.3 expressing N2 strain was designated strain UG1547.

An electropharyngogram of the human Kv4.3-expressing strain UG 1547 was recorded using an Axopatch-1D enhancer (Axon instruments) essentially as described in Raizen and Avery, supra; this one is in 1 shown. For comparison, reference is made to the EPGs for wild-type and exp-2 by Davis, Dent and Avery, Worm Breeders Gazette 14 (4): 72 (1996), and to EPGs for wild-type, Raizen and Avery.

From the comparison of the EPG of 1 With the EPG's for wild type in the above references, it can be seen that the expression of hKv4.3 at N2 compared to a significant reduction in time between the E and R peaks (the "P" phase) of the action potential ("AP") leads to the wild type, eg from a time in the range of 100-200 msec. in the wild type to a time of less than 50 msec. hKv4.3 expressing strain (eg, about 10-20 msec or less). This allows easy and convenient discrimination of "Kv4.3-like" APs from "wild-type-like" or "normal" APs in the recorded EPG.

Even though the invention is not limited to a specific explanation or hypothesis, It is believed that this reduction of the P phase by the functional expressed hKv4.3 channel is mediated, resulting in a functional Outflow of potassium ions leads. This is also confirmed by the example below.

Example XIII: Influence of 4-aminopyridine on the electropharyngogram at hKv4.3 expressing C elegans strain

Of the Influence of 4-aminopyridine on the EPG of an hKv4.3 expressing Strain was generated using the hKv4.3-expressing pha-1 strain UG 1598 of Example III. As mentioned in Example VI, the leads Exposure of the strain UG 1598 with 4-aminopyridine to a reduction pharyngeal pumping.

The UG 1598 EPGs (using 4 pharynxes) were recorded essentially as described in Example XII. The EPGs of UG 1598 without 4-aminopyridine were over recorded a period of 5 minutes, after which the pharynx 215 mM 4-aminopyridine were exposed, and the EPGs during further 5 minutes were recorded. The EPGs with and without presence of 4-aminopyridine were then combined with each other and with the wild-type EPG (see those mentioned in Example XII References) compared.

As expected, the EPGs showed for UG 1598 Action potentials (APs) with a significantly reduced P-phase similar to the wild type the reduction of P-phase in the APs, for the strain UG 1547 in Example XII was observed. The number of "Kv4.3-like" APs and the number of "normal" APs during the 5 minute Recording time was determined / counted from the recorded EPGs.

To Exposure to 4-aminopyridine was the number of "Kv4.3-like" APs and number the "normal" APs during the 5 minute Recording time, in turn, determined / counted from the recorded EPGs.

To Exposure to 4-aminopyridine was as compared to UG 1598 without 4-aminopyridine showed a significant decrease in the relative number of "Kv4.3-like" APs and a corresponding one Increase in the relative number of "normal" APs observed. In an experiment increased the number of "normal" APs during the 5 minute Recording time of 14 +/- 12 before the addition of 4-aminopyridine to 64 +/- 9 after the addition of 4-aminopyridine (The results are based on the recording of the EPGs of 4 pharyngeal cells; the paired student's T-test analysis (using Excel software) gives a P-value of P <0.0161). These results confirm among others, that the reduction of the P phase, as with the Kv4.3 expressing strains observed, indeed caused by the functionally expressed hKv4.3 proteins.

Example XIV: Screening a study group of CNS-active connections

One hKv4.3 expressing integrated C. elegans strain under control A myo-2 promoter in a wild-type background was used to create a Group of 203 commercially available Screen compounds with known CNS activity.

Of the parameter used was the drinking behavior, measured by the Uptake of Calcein AM over the time (see Example IX). The screen was automated with high throughput, where about 10,000 data points / day of screening were measured (for one general description of high-speed automated procedures Throughput for measuring the drinking behavior is among other things see WO 00/63427 and WO 00/63425 of the applicant).

From Of the 203 compounds tested, eight were inhibited drinking by> 60% Drinking inhibition and were using dose-response curves as hits approved. This corresponds to a hit rate of 3.9%.

SEQUENCE LISTING

Claims (18)

Roundworm (nematode), characterized in that the worm expresses a heterologous nucleotide sequence which codes for a functional voltage-dependent (potential-dependent) potassium channel of the Kv4 family, the Kv4.2 or Kv4.3 or a mutant thereof with a sequence identity of at least 50% represents the amino acid level with the corresponding human Kv4.2 or Kv4.3 channel. Nematode worm according to claim 1, characterized that the voltage-dependent The potassium channel encoding the Kv4 family nucleotide sequence by the SEQ ID NO 3 represents is. Nematode worm according to claim 2, characterized in that the nucleotide sequence encodes a mammalian Kv4 channel. Nematode worm according to one of claims 1 to 3, characterized that the voltage-dependent Potassium channel of the Kv4 family in at least the pharyngeal muscle, the body wall muscle and / or the vulva muscle is expressed. Nematode worm according to claim 4, characterized in that that the voltage-dependent Potassium channel of the Kv4 family expressed in a mutant nematode which is in an electropharynogram one compared to Wild type or reduced to N2 relaxation peak and / or a diminished refractory period shows. Nematode worm according to one of claims 1 to 5, characterized that the roundworm, after expression of the voltage-dependent potassium channel the Kv4 family, a change the muscle function, a change muscle contraction and / or relaxation, a change at the rate of pharyngeal pumping and / or a change in drinking, compared to the roundworm, which is used to express the voltage-gated potassium channel the Kv4 family is used. Nematode worm according to claim 6, wherein the roundworm after Expression of the voltage-dependent Potassium channels of the Kv4 family undergo a change in function, contraction and / or relaxation of the pharyngeal muscle. Nematode worm according to claim 7, wherein the change in the function of the pharyngeal muscle, a change in the polarization and / or Repolarization is what in the electropharynogram and / or at intracellular electrical Recording is observed. Nematode worm according to one of claims 1, 3 or 4, characterized that the threadworm is C. elegans UG1598 (LMBP 5851CB). Nematode worm according to one of claims 1 to 9, wherein the voltage-dependent Potassium channel of the Kv4 family with one or more auxiliary proteins, acceptor and / or subunits for voltage-gated potassium channels of the Kv4 family selected from KChiPs, including, but not limited to, KChiP1, KChiP2, KChiP3 and Kvβ, coexpressed is. Nematode worm according to claim 10, wherein the voltage-dependent potassium channel the Kv4 family with one or more Hilfsproteinen, acceptor molecules and / or Subunits for KChiP 2.2 is co-expressed. Use of a threadworm according to one of claims 1 to 11 to determine if a connection to the voltage-gated potassium channel the Kv4 family interacts. Method of identifying a compound those with a voltage-dependent Potassium channel of the Kv4 family can interact, the procedure the following steps include: (a) contacting one Nematode worm according to one of the claims 1 to 11 with the compound; and (b) detecting and / or observing, whether the compound has a detectable change in the nematode or in it causes; wherein the detectable change identifies a compound, which can interact with the Kv4 channel. Method of identifying a compound the prevention and / or treatment of a disease or disorder which is associated with a defect in a Kv4 channel is or is caused by, the process being the following Steps includes: (a) contacting a roundworm one of the claims 1 to 11 with the compound; and (b) detecting and / or observing, whether the compound has a detectable change in the nematode or in it causes; wherein the detectable change identifies a compound, the prevention and / or the treatment can be used. The method of claim 14, wherein the disease or disorder a heart disorder which is associated with or caused by a defect in a Kv4 channel is, and / or a disease or disorder of the nervous system that is associated with or caused by a defect in a Kv4 channel becomes. The method of claim 15, wherein the cardiac disorder, the is associated with or caused by a defect in a Kv4 channel is, arrhythmia is. The method of claim 15, wherein the disease or disorder of the nervous system is a disease or disorder of the central nervous system is. Method according to one of claims 13 to 17, wherein the at least a change, which is detected and / or observed in step (b), a change the muscle function, a change the neural function, a change in the rate of pharyngeal pumping, a change in drinking, a change the movement, a change when laying eggs and / or a change at the defecation is, compared to the roundworm, the voltage-dependent potassium channel of the Kv4 family before step (a).